+publisher:"University Utrecht" +contributor:("Heck, Albert")

1. Rozenbrand, J. Monolithic and Small Particle Column Materials for Application in Proteomics.

Degree: 2012, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/257922 ; URN:NBN:NL:UI:10-1874-257922 ; 1874/257922 ; urn:isbn:9789039358672 ; URN:NBN:NL:UI:10-1874-257922 ; https://dspace.library.uu.nl/handle/1874/257922

► In this thesis the influence of the capillary liquid chromatography separation on the identification of protein digests is studied. In the first part the chromatographic… (more)

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APA (6^th Edition):

Rozenbrand, J. (2012). Monolithic and Small Particle Column Materials for Application in Proteomics. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/257922 ; URN:NBN:NL:UI:10-1874-257922 ; 1874/257922 ; urn:isbn:9789039358672 ; URN:NBN:NL:UI:10-1874-257922 ; https://dspace.library.uu.nl/handle/1874/257922

Chicago Manual of Style (16^th Edition):

Rozenbrand, J. “Monolithic and Small Particle Column Materials for Application in Proteomics.” 2012. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/257922 ; URN:NBN:NL:UI:10-1874-257922 ; 1874/257922 ; urn:isbn:9789039358672 ; URN:NBN:NL:UI:10-1874-257922 ; https://dspace.library.uu.nl/handle/1874/257922.

MLA Handbook (7^th Edition):

Rozenbrand, J. “Monolithic and Small Particle Column Materials for Application in Proteomics.” 2012. Web. 22 Jan 2021.

Vancouver:

Rozenbrand J. Monolithic and Small Particle Column Materials for Application in Proteomics. [Internet] [Doctoral dissertation]. University Utrecht; 2012. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/257922 ; URN:NBN:NL:UI:10-1874-257922 ; 1874/257922 ; urn:isbn:9789039358672 ; URN:NBN:NL:UI:10-1874-257922 ; https://dspace.library.uu.nl/handle/1874/257922.

Council of Science Editors:

Rozenbrand J. Monolithic and Small Particle Column Materials for Application in Proteomics. [Doctoral Dissertation]. University Utrecht; 2012. Available from: https://dspace.library.uu.nl/handle/1874/257922 ; URN:NBN:NL:UI:10-1874-257922 ; 1874/257922 ; urn:isbn:9789039358672 ; URN:NBN:NL:UI:10-1874-257922 ; https://dspace.library.uu.nl/handle/1874/257922

2. Synowsky, S.A. Analysis of endogenous protein complexes by mass spectrometry.

Degree: 2008, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; 1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; https://dspace.library.uu.nl/handle/1874/27481

► Proteins are organized in large protein complexes that form an extensive network in the cell. They are the most versatile macromolecule in the cell and… (more)

▼ Proteins are organized in large protein complexes that form an extensive network in the cell. They are the most versatile macromolecule in the cell and the interactions between each other are highly directed and essential for most cellular functions. The activity of protein complexes is in turn frequently regulated by post-translational modifications such as acetylation, phosphorylation, etc. Since protein complexes determine to a large extend the functional organization of a cell, their investigation is of high importance to achieve a better understanding of cellular pathways and biological function. The analysis of endogenously expressed protein complexes is particularly interesting and relevant as it represents the most authentic situation in the cell. Proteins assemble with their natural interaction partner and are modulated according to a genuine situation. The introduction of tandem affinity purification procedures made it possible to isolate endogenous protein complexes on a large scale directly from cells. The procedure consists of two subsequent purification steps which keep the protein interactions intact throughout the procedure. The technique is relatively fast, reproducible and yields in pure protein complexes. By proteomics approaches large protein-protein interaction networks of S. cerevisiae have been unravelled which indicated that proteins are organized into modules of protein complexes. These proteomics experiments provided very useful information about the constituents of different protein assemblies. The next step towards a comprehensive analysis of protein assemblies is to characterize these protein assemblies in more detail. It is important to determine the direct interactions between proteins, the overall topology of protein complexes, strong and weak interacting proteins, subtle differences between closely related protein complexes and how protein interactions are modulated. We set out to describe the local network of protein complexes that are involved in RNA metabolism in the cell in detail and focus in particular on the nuclear and cytoplasmic exosome and the Ski complex. The exosome plays a crucial role in the degradation of mRNA and also in the processing of diverse RNA such as rRNA, snRNA and snoRNA. The cytoplasmic exosome requires the Ski complex as a co-activator for the degradation of mRNA. These endogenous heterogeneous proteins assemblies are characterized by a combination of tandem affinity purification technique and multiplexed mass spectrometry approaches. (Quantitative) proteomics approaches identified all exosome proteins and yielded in a first generation phosphorylation map with a relative quantitation of exosome proteins and identified phosphorylation sites between the nuclear and cytoplasmic variant. A strong emphasis is placed on native (tandem) mass spectrometry as it offers unique possibilities to investigate structural features of protein assemblies. The requirement of just a low amount of protein complex for a comprehensive mass spectrometry analysis makes this… Advisors/Committee Members: Heck, Albert.

Subjects/Keywords: Farmacie/Biofarmaceutische wetenschappen (FARM); Farmacie(FARM)

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APA (6^th Edition):

Synowsky, S. A. (2008). Analysis of endogenous protein complexes by mass spectrometry. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; 1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; https://dspace.library.uu.nl/handle/1874/27481

Chicago Manual of Style (16^th Edition):

Synowsky, S A. “Analysis of endogenous protein complexes by mass spectrometry.” 2008. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; 1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; https://dspace.library.uu.nl/handle/1874/27481.

MLA Handbook (7^th Edition):

Synowsky, S A. “Analysis of endogenous protein complexes by mass spectrometry.” 2008. Web. 22 Jan 2021.

Vancouver:

Synowsky SA. Analysis of endogenous protein complexes by mass spectrometry. [Internet] [Doctoral dissertation]. University Utrecht; 2008. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; 1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; https://dspace.library.uu.nl/handle/1874/27481.

Council of Science Editors:

Synowsky SA. Analysis of endogenous protein complexes by mass spectrometry. [Doctoral Dissertation]. University Utrecht; 2008. Available from: https://dspace.library.uu.nl/handle/1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; 1874/27481 ; URN:NBN:NL:UI:10-1874-27481 ; https://dspace.library.uu.nl/handle/1874/27481

3. Lorenzen, K. From structure to function : Protein assemblies dissected by mass spectrometry.

Degree: 2008, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/31369 ; URN:NBN:NL:UI:10-1874-31369 ; 1874/31369 ; urn:isbn:9789039349304 ; URN:NBN:NL:UI:10-1874-31369 ; https://dspace.library.uu.nl/handle/1874/31369

► This thesis demonstrates some of the possibilities mass spectrometry can provide to gain new insight into structure and function of protein complexes. While technologies in… (more)

▼ This thesis demonstrates some of the possibilities mass spectrometry can provide to gain new insight into structure and function of protein complexes. While technologies in native mass spectrometry are still under development, it already allows research on complete proteins and protein complexes up to a seemingly unlimited size. This would not have been possible without the technical developments in all related fields, for example ionization, instrumentation and sample preparation and handling. An example for further instrumentation development is given in chapter two of this thesis. Here we attempted to improve one of the instruments frequently used in native mass spectrometry, the Q ToF, by modifying the pressures, nature of collision gas and orifi ces of the collision cell. These changes on the Q ToF allowed us to obtain the results that are described in chapter 2 to 4 of this thesis. Nowadays mass spectrometry aims to bridge between data of high throughput proteomic screens of protein networks in the cell and high resolution structural data obtained by methods like x ray, cryo EM and NMR. The most direct result obtained by native mass spectrometry for protein complexes is often the stoichiometry. While with homogeneous complexes this is visible at fi rst sight most of the times, heterogeneous complexes often need to be dissociated by tandem mass spectrometry. In chapter 3 we determined the stoichiometry of the purifi ed portal ring and the assembly of the portal ring with the fi rst tail accessory factor gp4 of bacteriophage P22. Besides determining the stoichiometry we were also able to make conclusions about the binding behavior and structural changes that occur upon the assembly. Charge state distributions generated in the ESI process arouse the suspicion that the portal might undergo a major conformational change upon binding of gp4. A new technique called ion mobility mass spectrometry confi rmed this structural change. These results gave new insights into the maturation process of P22. Several interactome studies have shown that it is possible to establish interaction networks of endogenously expressed proteins in yeast by affi nity pulldowns combined with mass spectrometry. While these studies have shown that proteins almost always work in interaction with other proteins they fail to give information about the structure and stoichiometry of the proteins. Native mass spectrometry can provide new and complementary information here. We have analyzed the architecture of the multisubunit RNA polymerases Pol II and Pol III. The measurements presented in chapter 4 resulted in insights into the subunit architecture of Pol II and III. We here compared the results gained from Pol II, where detailed structural properties are known, with Pol III. The data demonstrates that each of the 17 subunits of Pol III is present as a single copy. Our analysis provided new information about the heterodimeric subcomplexes in Pol III. The obtained model for the Pol III subunit architecture might guide future biochemical and… Advisors/Committee Members: Heck, Albert.

Subjects/Keywords: Farmacie/Biofarmaceutische wetenschappen (FARM); Farmacie(FARM)

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APA (6^th Edition):

Lorenzen, K. (2008). From structure to function : Protein assemblies dissected by mass spectrometry. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/31369 ; URN:NBN:NL:UI:10-1874-31369 ; 1874/31369 ; urn:isbn:9789039349304 ; URN:NBN:NL:UI:10-1874-31369 ; https://dspace.library.uu.nl/handle/1874/31369

Chicago Manual of Style (16^th Edition):

Lorenzen, K. “From structure to function : Protein assemblies dissected by mass spectrometry.” 2008. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/31369 ; URN:NBN:NL:UI:10-1874-31369 ; 1874/31369 ; urn:isbn:9789039349304 ; URN:NBN:NL:UI:10-1874-31369 ; https://dspace.library.uu.nl/handle/1874/31369.

MLA Handbook (7^th Edition):

Lorenzen, K. “From structure to function : Protein assemblies dissected by mass spectrometry.” 2008. Web. 22 Jan 2021.

Vancouver:

Lorenzen K. From structure to function : Protein assemblies dissected by mass spectrometry. [Internet] [Doctoral dissertation]. University Utrecht; 2008. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/31369 ; URN:NBN:NL:UI:10-1874-31369 ; 1874/31369 ; urn:isbn:9789039349304 ; URN:NBN:NL:UI:10-1874-31369 ; https://dspace.library.uu.nl/handle/1874/31369.

Council of Science Editors:

Lorenzen K. From structure to function : Protein assemblies dissected by mass spectrometry. [Doctoral Dissertation]. University Utrecht; 2008. Available from: https://dspace.library.uu.nl/handle/1874/31369 ; URN:NBN:NL:UI:10-1874-31369 ; 1874/31369 ; urn:isbn:9789039349304 ; URN:NBN:NL:UI:10-1874-31369 ; https://dspace.library.uu.nl/handle/1874/31369

4. Dadvar, P. Probing the drug interactome by chemical proteomics.

Degree: 2009, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; 1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; https://dspace.library.uu.nl/handle/1874/36536

► Approved PDE5 inhibitors for the treatment of erectile dysfunction (ED) include sildenafil (Viagra), vardenafil (Levitra) and tadalafil (Cialis), all of which are considered very specific… (more)

Subjects/Keywords: Farmacie/Biofarmaceutische wetenschappen (FARM); Farmacie(FARM)

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APA (6^th Edition):

Dadvar, P. (2009). Probing the drug interactome by chemical proteomics. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; 1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; https://dspace.library.uu.nl/handle/1874/36536

Chicago Manual of Style (16^th Edition):

Dadvar, P. “Probing the drug interactome by chemical proteomics.” 2009. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; 1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; https://dspace.library.uu.nl/handle/1874/36536.

MLA Handbook (7^th Edition):

Dadvar, P. “Probing the drug interactome by chemical proteomics.” 2009. Web. 22 Jan 2021.

Vancouver:

Dadvar P. Probing the drug interactome by chemical proteomics. [Internet] [Doctoral dissertation]. University Utrecht; 2009. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; 1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; https://dspace.library.uu.nl/handle/1874/36536.

Council of Science Editors:

Dadvar P. Probing the drug interactome by chemical proteomics. [Doctoral Dissertation]. University Utrecht; 2009. Available from: https://dspace.library.uu.nl/handle/1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; 1874/36536 ; URN:NBN:NL:UI:10-1874-36536 ; https://dspace.library.uu.nl/handle/1874/36536

5. Uetrecht, C. Analysing macromolecular structures with native mass spectrometry : insights to virus structure and assembly.

Degree: 2010, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/187998 ; URN:NBN:NL:UI:10-1874-187998 ; 1874/187998 ; urn:isbn:9789039354421 ; URN:NBN:NL:UI:10-1874-187998 ; https://dspace.library.uu.nl/handle/1874/187998

► In the past years, native mass spectrometry (MS) has gained attention in research and also industry. The technique can provide information on mass, topology, binding… (more)

▼ In the past years, native mass spectrometry (MS) has gained attention in research and also industry. The technique can provide information on mass, topology, binding affinity or stability and dynamics of macromolecular structures. With ion mobility (IM) even conformational changes can be monitored. With this technique, viral protein complexes involved in capsid formation were analysed. These nanocontainers enclose the genome in viruses. Even though studied for decades, many questions remain, for example, whether the capsid assembly proceeds to completion. For Hepatitis B virus (HBV), charge state distributions could be resolved for the two capsid species. The assigned masses of ~ 3 and 4 MDa (standard deviation below 0.1%) were in agreement with completely assembled capsids consisting of 90 or 120 dimers as also confirmed by collision induced dissociation (CID). The two HBV capsids have similar internal energies in the gas phase enabling a direct comparison of their stability in CID. Interestingly, the smaller assembly exhibited a higher resistance to CID in agreement with previous mutagenesis studies. However, the mechanical properties of the two geometries were similar in atomic force microscopy (AFM) enforcing that the relation between gas phase and in solution stability is not trivial. Furthermore, theoretical models predicted exchange of dimers between capsids and free ones in solution. I monitored the dynamic incorporation of isotopically labelled dimers into preassembled HBV capsids using CID on selected species. A slow exchange occurred over months exclusively at low temperatures and independent of solution pH, but only in the smaller capsids. IMMS provides structural information at low resolution and is often combined with computational modelling. The question arises to which degree structures are preserved in the gas phase. For the large viral assemblies, shape determination in IMMS was possible with some adjustments. Remarkably, the HBV capsids exhibit a vacuum shape consistent with the hollow spheres observed in electron microscopy or AFM and with dimensions modelled for the two species. Of special interest to vaccine and drug development are viral assembly and disassembly pathways. We found that the norovirus, causing viral gastroenteritis, shows a complex albeit reversible dissociation and reassociation behaviour dependent on pH and ionic strength. The detected oligomers including a 60-mer, an 80-mer and the 180-mer capsid likely interconverted via dimers. Surprisingly, the 60-mer and 80-mer also resembled hollow capsid-like structures in IMMS and AFM. The extraordinarily high sensitivity in IMMS enabled studies on norovirus and also HBV oligomers, which are inaccessible by common techniques in structural biology. Comparison of data on the viral intermediates and globular protein complexes as well as modelled results indicated that the intermediates have extended structures as in assembled capsids. Further analysis provided insights into the priming of and a common pathway for assembly in both viruses.… Advisors/Committee Members: Heck, Albert.

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APA (6^th Edition):

Uetrecht, C. (2010). Analysing macromolecular structures with native mass spectrometry : insights to virus structure and assembly. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/187998 ; URN:NBN:NL:UI:10-1874-187998 ; 1874/187998 ; urn:isbn:9789039354421 ; URN:NBN:NL:UI:10-1874-187998 ; https://dspace.library.uu.nl/handle/1874/187998

Chicago Manual of Style (16^th Edition):

Uetrecht, C. “Analysing macromolecular structures with native mass spectrometry : insights to virus structure and assembly.” 2010. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/187998 ; URN:NBN:NL:UI:10-1874-187998 ; 1874/187998 ; urn:isbn:9789039354421 ; URN:NBN:NL:UI:10-1874-187998 ; https://dspace.library.uu.nl/handle/1874/187998.

MLA Handbook (7^th Edition):

Uetrecht, C. “Analysing macromolecular structures with native mass spectrometry : insights to virus structure and assembly.” 2010. Web. 22 Jan 2021.

Vancouver:

Uetrecht C. Analysing macromolecular structures with native mass spectrometry : insights to virus structure and assembly. [Internet] [Doctoral dissertation]. University Utrecht; 2010. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/187998 ; URN:NBN:NL:UI:10-1874-187998 ; 1874/187998 ; urn:isbn:9789039354421 ; URN:NBN:NL:UI:10-1874-187998 ; https://dspace.library.uu.nl/handle/1874/187998.

Council of Science Editors:

Uetrecht C. Analysing macromolecular structures with native mass spectrometry : insights to virus structure and assembly. [Doctoral Dissertation]. University Utrecht; 2010. Available from: https://dspace.library.uu.nl/handle/1874/187998 ; URN:NBN:NL:UI:10-1874-187998 ; 1874/187998 ; urn:isbn:9789039354421 ; URN:NBN:NL:UI:10-1874-187998 ; https://dspace.library.uu.nl/handle/1874/187998

6. Mischerikow, N. Proteomics of transcription factors.

Degree: 2011, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/205238 ; URN:NBN:NL:UI:10-1874-205238 ; 1874/205238 ; urn:isbn:9789064644641 ; URN:NBN:NL:UI:10-1874-205238 ; https://dspace.library.uu.nl/handle/1874/205238

► Peptide mass spectrometry (MS) is an invaluable analytical method in biological and medical research. It is the only technique that, when integrated with liquid chromatography… (more)

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APA (6^th Edition):

Mischerikow, N. (2011). Proteomics of transcription factors. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/205238 ; URN:NBN:NL:UI:10-1874-205238 ; 1874/205238 ; urn:isbn:9789064644641 ; URN:NBN:NL:UI:10-1874-205238 ; https://dspace.library.uu.nl/handle/1874/205238

Chicago Manual of Style (16^th Edition):

Mischerikow, N. “Proteomics of transcription factors.” 2011. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/205238 ; URN:NBN:NL:UI:10-1874-205238 ; 1874/205238 ; urn:isbn:9789064644641 ; URN:NBN:NL:UI:10-1874-205238 ; https://dspace.library.uu.nl/handle/1874/205238.

MLA Handbook (7^th Edition):

Mischerikow, N. “Proteomics of transcription factors.” 2011. Web. 22 Jan 2021.

Vancouver:

Mischerikow N. Proteomics of transcription factors. [Internet] [Doctoral dissertation]. University Utrecht; 2011. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/205238 ; URN:NBN:NL:UI:10-1874-205238 ; 1874/205238 ; urn:isbn:9789064644641 ; URN:NBN:NL:UI:10-1874-205238 ; https://dspace.library.uu.nl/handle/1874/205238.

Council of Science Editors:

Mischerikow N. Proteomics of transcription factors. [Doctoral Dissertation]. University Utrecht; 2011. Available from: https://dspace.library.uu.nl/handle/1874/205238 ; URN:NBN:NL:UI:10-1874-205238 ; 1874/205238 ; urn:isbn:9789064644641 ; URN:NBN:NL:UI:10-1874-205238 ; https://dspace.library.uu.nl/handle/1874/205238

7. Bereszczak, J.Z. Probing structure, stability and dynamics of viruses by mass spectrometry.

Degree: 2014, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; 1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; https://dspace.library.uu.nl/handle/1874/289085

► Our capacity to deal with many viral infections has vastly improved, largely through the development of successful vaccines and antivirals. However, for some viruses and… (more)

▼ Our capacity to deal with many viral infections has vastly improved, largely through the development of successful vaccines and antivirals. However, for some viruses and through the emergence of new strains and more resistant strains, this has proved more challenging. By continuing to explore the fundamental processes underlying viruses and their interaction with their host, it may be possible to treat or prevent these more challenging viral infections. In this thesis I probe the structure, stability and dynamics of viruses by mass spectrometry (MS), more specifically I explore these aspects for a self-assembling virus-like particle (VLP), the P particle, that has the potential as a chimeric vaccine and for the hepatitis B virus (HBV) and the HBV related e-antigen. The stability and dynamics of the P particle, important properties that would have implications in its use as a therapeutic vaccine, were investigated. We characterised the P particle under reducing and non-reducing conditions, due to the presence of inter-subunit disulphide bonds, and identified previously uncharacterised P domain complexes. By changing the solution phase pH and ionic strength we showed how the equilibrium between these complexes could be manipulated. Using ion mobility mass spectrometry (IMMS) in combination with molecular modeling we were able to propose probable structures for these P domain complexes. Our results demonstrate the interchangeable nature and dynamic relationship of all P domain complexes and confirm their binding activity to the host receptors. The interaction of the HBV capsid with two antibodies, Fab E1 and Fab 3120, that bind through distinct epitopes was studied. We used an approach of hydrogen deuterium exchange-mass spectrometry (HDX-MS) to examine the dynamics of the interaction, identifying a reduction in deuterium incorporation both at the binding epitope on HBV and regions distal to the epitope. Overall our results suggest a global rigidification of the capsid and suppression of its breathing motions. We employed an alternative methodology to further characterise the interaction, this time employing two complementary ESI-based techniques, native MS and gas-phase electrophoretic mobility molecular analysis (GEMMA). We demonstrate their capability to monitor the concentration-dependant antibody binding process and their ability to resolve different modes of antibody binding to the T = 3 and T = 4 capsid morphologies, consistent with the cryo-EM model of binding. These data indicate a rapid means of characterisation of such complexes. We further applied the HDX-MS methodology to study the structural properties of the HBV core-antigen (cAg) and the e-antigen (eAg) (an immune regulator). Despite almost complete sequence identity, the dimeric proteins adopt distinct quaternary structures, the likely basis of their differing properties. HDX-MS detected many regions that differed substantially in their HDX dynamics. Comparison of the HDX of unassembled cAg with that in assembled capsids indicated increased resistance… Advisors/Committee Members: Heck, Albert.

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APA (6^th Edition):

Bereszczak, J. Z. (2014). Probing structure, stability and dynamics of viruses by mass spectrometry. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; 1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; https://dspace.library.uu.nl/handle/1874/289085

Chicago Manual of Style (16^th Edition):

Bereszczak, J Z. “Probing structure, stability and dynamics of viruses by mass spectrometry.” 2014. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; 1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; https://dspace.library.uu.nl/handle/1874/289085.

MLA Handbook (7^th Edition):

Bereszczak, J Z. “Probing structure, stability and dynamics of viruses by mass spectrometry.” 2014. Web. 22 Jan 2021.

Vancouver:

Bereszczak JZ. Probing structure, stability and dynamics of viruses by mass spectrometry. [Internet] [Doctoral dissertation]. University Utrecht; 2014. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; 1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; https://dspace.library.uu.nl/handle/1874/289085.

Council of Science Editors:

Bereszczak JZ. Probing structure, stability and dynamics of viruses by mass spectrometry. [Doctoral Dissertation]. University Utrecht; 2014. Available from: https://dspace.library.uu.nl/handle/1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; 1874/289085 ; URN:NBN:NL:UI:10-1874-289085 ; https://dspace.library.uu.nl/handle/1874/289085

8. Giansanti, Piero. Signaling network dynamics investigated by quantitative phosphoproteomics.

Degree: 2014, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/308082 ; URN:NBN:NL:UI:10-1874-308082 ; 1874/308082 ; urn:isbn:9789088919824 ; URN:NBN:NL:UI:10-1874-308082 ; https://dspace.library.uu.nl/handle/1874/308082

► This thesis describes the application of proteomics technologies to get insight into several aspects of phosphorylation signaling dynamics. The core tool in all performed experiments… (more)

▼ This thesis describes the application of proteomics technologies to get insight into several aspects of phosphorylation signaling dynamics. The core tool in all performed experiments is mass spectrometry (MS)-based phosphoproteomics. In Chapter 1, a general introduction is given into proteomics and MS-based proteomics workflows. In Chapter 2, we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the PKA consensus phosphorylation motif [R/K][R/K/X]X[pS/pT]. This targeted phosphoproteomic strategy, in combination with stable isotope dimethyl labeling, is used to profile temporal changes of potential PKA substrates in Jurkat T lymphocytes upon PGE2 stimulation, which increases intracellular cAMP, thereby activating PKA. It is shown that this approach is very specific and highly complementary to a large-scale phosphoproteomics approach, enabling to profile hundreds of putative PKA sites. In Chapter 3, the phosphopeptide enrichment robustness of Ti4+-IMAC is evaluated. First, we prove that Ti4+-IMAC enrichment allows a highly reproducible quantification of phosphorylation sites in HeLa cells. Subsequently, we apply this strategy to monitor the phosphoproteome of Jurkat T lymphocytes upon PGE2 stimulation, covering extended time series. We demonstrate that this enrichment strategy in combination with label-free quantification enables in-depth investigation of phosphorylation dynamics, highlighting differential regulation of different kinases over time. In Chapter 4, we employ a three-pronged MS-based proteomics strategy to identify the direct targets and downstream signaling effect of four tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib) in A431 epidermoid carcinoma cells, as a model system for skin-cancer. The integration of chemical proteomics and phosphoproteomics allows us to define a set of signaling nodes modulated by each individual drug that can be considered putative targets in epithelial cancers. In Chapter 5, we assess the beneficial use of complementary proteases, namely trypsin, LysC, GluC, AspN and chymotrypsin for the phosphoproteome analysis of Jurkat T cells, analyzed by using Ti4+-IMAC enrichment. The obtained results are a significant improvement upon data from a single protease digest. We demonstrate that nearly each phosphosite can be linked to a preferred protease forming detectable phosphopeptides, whereby the gain in using alternative proteases can be more than 10,000 fold for specific sites in intensity. Moreover, many of the identified sites are not yet reported in currently available and public depositories, demonstrating the complementary nature of these enzymes, through which different parts of the phosphoproteome can be uncovered. Advisors/Committee Members: Heck, Albert.

Subjects/Keywords: protein phosphorylation; quantitative phosphoproteomics; mass spectrometry; signaling network; kinases; tyrosine kinases inhibitors

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APA (6^th Edition):

Giansanti, P. (2014). Signaling network dynamics investigated by quantitative phosphoproteomics. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/308082 ; URN:NBN:NL:UI:10-1874-308082 ; 1874/308082 ; urn:isbn:9789088919824 ; URN:NBN:NL:UI:10-1874-308082 ; https://dspace.library.uu.nl/handle/1874/308082

Chicago Manual of Style (16^th Edition):

Giansanti, Piero. “Signaling network dynamics investigated by quantitative phosphoproteomics.” 2014. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/308082 ; URN:NBN:NL:UI:10-1874-308082 ; 1874/308082 ; urn:isbn:9789088919824 ; URN:NBN:NL:UI:10-1874-308082 ; https://dspace.library.uu.nl/handle/1874/308082.

MLA Handbook (7^th Edition):

Giansanti, Piero. “Signaling network dynamics investigated by quantitative phosphoproteomics.” 2014. Web. 22 Jan 2021.

Vancouver:

Giansanti P. Signaling network dynamics investigated by quantitative phosphoproteomics. [Internet] [Doctoral dissertation]. University Utrecht; 2014. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/308082 ; URN:NBN:NL:UI:10-1874-308082 ; 1874/308082 ; urn:isbn:9789088919824 ; URN:NBN:NL:UI:10-1874-308082 ; https://dspace.library.uu.nl/handle/1874/308082.

Council of Science Editors:

Giansanti P. Signaling network dynamics investigated by quantitative phosphoproteomics. [Doctoral Dissertation]. University Utrecht; 2014. Available from: https://dspace.library.uu.nl/handle/1874/308082 ; URN:NBN:NL:UI:10-1874-308082 ; 1874/308082 ; urn:isbn:9789088919824 ; URN:NBN:NL:UI:10-1874-308082 ; https://dspace.library.uu.nl/handle/1874/308082

9. Snijder, J. Extending the boundaries of native mass spectrometry to study virus structure and assembly.

Degree: 2015, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/319575 ; URN:NBN:NL:UI:10-1874-319575 ; 1874/319575 ; urn:isbn:9789462952720 ; URN:NBN:NL:UI:10-1874-319575 ; https://dspace.library.uu.nl/handle/1874/319575

► Native mass spectrometry (MS) is a powerful tool to study the composition and quaternary structure of protein complexes over a wide range of size and… (more)

▼ Native mass spectrometry (MS) is a powerful tool to study the composition and quaternary structure of protein complexes over a wide range of size and mass. As an analytical tool, native MS offers unmatched specificity and precision to pinpoint the stoichiometry of biomolecular complexes. It has been developed for analysis of larger protein complexes, such as intact virus particles, in recent years. The use of native MS was shown to be particularly convenient to characterize the assembly and composition of these large particles, offering important information on their role as pathogens and their use for nanotechnology and medicine. This thesis describes the development and application of native MS to analyze the structure and assembly of virus particles. It is shown that virus particles as big as 18 megadalton can be analyzed with native MS on a modified quadrupole time-of-flight instrument. Analysis of the obtained peak shapes and theoretical considerations about the inherent spread in masses and the performance of the mass analyzer lead to the conclusion that the 18 megadalton particles are at the limit of what is feasible on the current generation of instruments, and shows that poor desolvation of the ions is currently the main limiting factor. Having established that 18 megadalton capsids can be analyzed by native MS, several maturation intermediates of bacteriophage HK97 were analyzed by native MS to learn about the maturation pathway of the virus. It is furthermore shown how we extended the mass range of a novel type of Orbitrap mass analyzer for native MS to allow analysis of virus particles of several megadaltons. The transmission at high mass-to-charge ratio is improved by modifying the ion guides of the instrument. Sensitivity is improved 5-fold in the high m/z range to allow analysis of virus particles with improved resolution compared to the time-of-flight based platforms. It is demonstrated that the Orbitrap with extended mass range can be used to quantify cargo loading in a virus-like particle for nanotechnology and that it can be used to determine the stoichiometry of capsids in heterogeneous mixtures. The use of both the quadrupole time-of-flight and the Orbitrap-based instrument is demonstrated in this thesis in three applications to virus structure and assembly: native mass spectrometry is used in combination with proteomics-type LC-MS/MS, hydrogen-deuterium exchange MS and atomic force microscopy to uncover a novel component of mature adenovirus, to study assembly and uncoating of triatoma virus and to study the effects of cargo encapsulation in a virus-like bacterial nanocompartment called encapsulin. This thesis thereby illustrates the great potential of native MS to study virus capsid structure and assembly. Several potential improvements of current native MS instrumentation as well as possible fundamental limitations of the technique are discussed to establish the current and future role of native MS in the analytical toolbox to study protein complex structure and assembly. Advisors/Committee Members: Heck, Albert.

Subjects/Keywords: mass spectrometry; virus; capsid; virus assembly; nano electrospray ionization

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APA (6^th Edition):

Snijder, J. (2015). Extending the boundaries of native mass spectrometry to study virus structure and assembly. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/319575 ; URN:NBN:NL:UI:10-1874-319575 ; 1874/319575 ; urn:isbn:9789462952720 ; URN:NBN:NL:UI:10-1874-319575 ; https://dspace.library.uu.nl/handle/1874/319575

Chicago Manual of Style (16^th Edition):

Snijder, J. “Extending the boundaries of native mass spectrometry to study virus structure and assembly.” 2015. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/319575 ; URN:NBN:NL:UI:10-1874-319575 ; 1874/319575 ; urn:isbn:9789462952720 ; URN:NBN:NL:UI:10-1874-319575 ; https://dspace.library.uu.nl/handle/1874/319575.

MLA Handbook (7^th Edition):

Snijder, J. “Extending the boundaries of native mass spectrometry to study virus structure and assembly.” 2015. Web. 22 Jan 2021.

Vancouver:

Snijder J. Extending the boundaries of native mass spectrometry to study virus structure and assembly. [Internet] [Doctoral dissertation]. University Utrecht; 2015. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/319575 ; URN:NBN:NL:UI:10-1874-319575 ; 1874/319575 ; urn:isbn:9789462952720 ; URN:NBN:NL:UI:10-1874-319575 ; https://dspace.library.uu.nl/handle/1874/319575.

Council of Science Editors:

Snijder J. Extending the boundaries of native mass spectrometry to study virus structure and assembly. [Doctoral Dissertation]. University Utrecht; 2015. Available from: https://dspace.library.uu.nl/handle/1874/319575 ; URN:NBN:NL:UI:10-1874-319575 ; 1874/319575 ; urn:isbn:9789462952720 ; URN:NBN:NL:UI:10-1874-319575 ; https://dspace.library.uu.nl/handle/1874/319575

10. Lössl, P. Integrative Mass Spectrometry Approaches to Monitor Protein Structures, Modifications, and Interactions.

Degree: 2017, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/351525 ; URN:NBN:NL:UI:10-1874-351525 ; 1874/351525 ; urn:isbn:9789039367636 ; URN:NBN:NL:UI:10-1874-351525 ; https://dspace.library.uu.nl/handle/1874/351525

► This thesis illustrates the current standing of mass spectrometry (MS) in molecular and structural biology. The primary aim of the herein described research is to… (more)

▼ This thesis illustrates the current standing of mass spectrometry (MS) in molecular and structural biology. The primary aim of the herein described research is to facilitate protein characterization by combining mass spectrometric methods among each other and with complementary analytical strategies. In Chapter 1, an overview of mass spectrometric methods that are most useful for studying the organization of protein assemblies is provided. Based on recent examples from literature, it illustrated how these MS methods can be integrated with other molecular and structural biology techniques. In Chapter 2, factors confining the obtainable mass resolution in native MS are identified. It is shown that mass resolution is mainly limited by inefficient analyte desolvation, i.e., the incomplete disruption of unwanted non-covalent analyte-small molecule adducts. It is furthermore discussed that a higher mass resolution is primarily required for native MS analysis of species with very small mass differences, e.g., protein complexes carrying different kinds and numbers of post-translational modifications. In Chapter 3, the benefits of high-resolution native MS are demonstrated by studying phosphorylation reactions in binary protein-ligand and protein-protein systems up to 155 kDa. Complementation of high-resolution native MS with bottom-up and top-down proteomics is shown to enable the characterization of enzymatic protein phosphorylation with respect to concurring non-covalent interactions, overall reaction kinetics and the sequence and completeness of residue-specific modification events. In Chapter 4, the scope of this approach is further extended by inclusion of cross-linking MS and ion mobility spectrometry MS to analyze the interdependence of multisite phosphorylation, interaction dynamics and conformational changes during the interplay of Polo-like kinase 1, Aurora kinase A and Bora. Uniting the data from all applied MS methods yields a mechanistic model of this three-protein reaction, illustrating that integrative MS-based approaches can provide a dynamic view on protein structures and interactions. This analytical angle is complementary to X-ray crystallography and cryo-electron microscopy data, suggesting a unique niche of MS in structural biology. In Chapter 5, potential functions of MS in hybrid structural biology approaches are further investigated. The analyses of the LRP1-RAP and KaiCBA complexes illustrate the value of complementing traditional biochemical and biophysical techniques with native MS to assess assembly states as well as condition-dependent association and dissociation processes. Both studies also show that cross-linking MS renders valuable insights into protein conformations and binding interfaces, especially when high-resolution structural characterization is not feasible. In all, it is demonstrated that MS is able to illuminate various facets of protein structures, modifications and interactions. Therefore, MS will continue to play a vital role in exploring the structural and chemical principles of… Advisors/Committee Members: Heck, Albert.

Subjects/Keywords: structural biology; post-translational modifications; protein complexes; mass spectrometry; proteomics

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APA (6^th Edition):

Lössl, P. (2017). Integrative Mass Spectrometry Approaches to Monitor Protein Structures, Modifications, and Interactions. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/351525 ; URN:NBN:NL:UI:10-1874-351525 ; 1874/351525 ; urn:isbn:9789039367636 ; URN:NBN:NL:UI:10-1874-351525 ; https://dspace.library.uu.nl/handle/1874/351525

Chicago Manual of Style (16^th Edition):

Lössl, P. “Integrative Mass Spectrometry Approaches to Monitor Protein Structures, Modifications, and Interactions.” 2017. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/351525 ; URN:NBN:NL:UI:10-1874-351525 ; 1874/351525 ; urn:isbn:9789039367636 ; URN:NBN:NL:UI:10-1874-351525 ; https://dspace.library.uu.nl/handle/1874/351525.

MLA Handbook (7^th Edition):

Lössl, P. “Integrative Mass Spectrometry Approaches to Monitor Protein Structures, Modifications, and Interactions.” 2017. Web. 22 Jan 2021.

Vancouver:

Lössl P. Integrative Mass Spectrometry Approaches to Monitor Protein Structures, Modifications, and Interactions. [Internet] [Doctoral dissertation]. University Utrecht; 2017. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/351525 ; URN:NBN:NL:UI:10-1874-351525 ; 1874/351525 ; urn:isbn:9789039367636 ; URN:NBN:NL:UI:10-1874-351525 ; https://dspace.library.uu.nl/handle/1874/351525.

Council of Science Editors:

Lössl P. Integrative Mass Spectrometry Approaches to Monitor Protein Structures, Modifications, and Interactions. [Doctoral Dissertation]. University Utrecht; 2017. Available from: https://dspace.library.uu.nl/handle/1874/351525 ; URN:NBN:NL:UI:10-1874-351525 ; 1874/351525 ; urn:isbn:9789039367636 ; URN:NBN:NL:UI:10-1874-351525 ; https://dspace.library.uu.nl/handle/1874/351525

11. Yang, Y. Exploring the sweetness of protein heterogeneity by hybrid mass spectrometry approaches.

Degree: 2017, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; 1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; https://dspace.library.uu.nl/handle/1874/354035

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Yang, Y. (2017). Exploring the sweetness of protein heterogeneity by hybrid mass spectrometry approaches. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; 1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; https://dspace.library.uu.nl/handle/1874/354035

Chicago Manual of Style (16^th Edition):

Yang, Y. “Exploring the sweetness of protein heterogeneity by hybrid mass spectrometry approaches.” 2017. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; 1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; https://dspace.library.uu.nl/handle/1874/354035.

MLA Handbook (7^th Edition):

Yang, Y. “Exploring the sweetness of protein heterogeneity by hybrid mass spectrometry approaches.” 2017. Web. 22 Jan 2021.

Vancouver:

Yang Y. Exploring the sweetness of protein heterogeneity by hybrid mass spectrometry approaches. [Internet] [Doctoral dissertation]. University Utrecht; 2017. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; 1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; https://dspace.library.uu.nl/handle/1874/354035.

Council of Science Editors:

Yang Y. Exploring the sweetness of protein heterogeneity by hybrid mass spectrometry approaches. [Doctoral Dissertation]. University Utrecht; 2017. Available from: https://dspace.library.uu.nl/handle/1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; 1874/354035 ; URN:NBN:NL:UI:10-1874-354035 ; https://dspace.library.uu.nl/handle/1874/354035

12. Čaval, Tomislav. Mass Spectrometric Exploration of the GlycoUniverse.

Degree: 2019, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/386312 ; URN:NBN:NL:UI:10-1874-386312 ; 1874/386312 ; urn:isbn:9789039372104 ; URN:NBN:NL:UI:10-1874-386312 ; https://dspace.library.uu.nl/handle/1874/386312

► Currently over 80% of biological drugs on the market are modified by sugar residues called glycans. Presence of these glycans on each of these molecules… (more)

Subjects/Keywords: Native mass spectrometry; Glycoproteomics; Glycosylation; Biotherapeutics; Glycoengineering

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APA (6^th Edition):

Čaval, T. (2019). Mass Spectrometric Exploration of the GlycoUniverse. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/386312 ; URN:NBN:NL:UI:10-1874-386312 ; 1874/386312 ; urn:isbn:9789039372104 ; URN:NBN:NL:UI:10-1874-386312 ; https://dspace.library.uu.nl/handle/1874/386312

Chicago Manual of Style (16^th Edition):

Čaval, Tomislav. “Mass Spectrometric Exploration of the GlycoUniverse.” 2019. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/386312 ; URN:NBN:NL:UI:10-1874-386312 ; 1874/386312 ; urn:isbn:9789039372104 ; URN:NBN:NL:UI:10-1874-386312 ; https://dspace.library.uu.nl/handle/1874/386312.

MLA Handbook (7^th Edition):

Čaval, Tomislav. “Mass Spectrometric Exploration of the GlycoUniverse.” 2019. Web. 22 Jan 2021.

Vancouver:

Čaval T. Mass Spectrometric Exploration of the GlycoUniverse. [Internet] [Doctoral dissertation]. University Utrecht; 2019. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/386312 ; URN:NBN:NL:UI:10-1874-386312 ; 1874/386312 ; urn:isbn:9789039372104 ; URN:NBN:NL:UI:10-1874-386312 ; https://dspace.library.uu.nl/handle/1874/386312.

Council of Science Editors:

Čaval T. Mass Spectrometric Exploration of the GlycoUniverse. [Doctoral Dissertation]. University Utrecht; 2019. Available from: https://dspace.library.uu.nl/handle/1874/386312 ; URN:NBN:NL:UI:10-1874-386312 ; 1874/386312 ; urn:isbn:9789039372104 ; URN:NBN:NL:UI:10-1874-386312 ; https://dspace.library.uu.nl/handle/1874/386312

13. Di Palma, S. Optimizing hydrophilic interaction liquid chromatography for ultrasensitive proteome analysis.

Degree: 2013, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/261028 ; URN:NBN:NL:UI:10-1874-261028 ; 1874/261028 ; urn:isbn:9789088915581 ; URN:NBN:NL:UI:10-1874-261028 ; https://dspace.library.uu.nl/handle/1874/261028

► In the last decade, the field of proteomics has rapidly progressed with substantial advances in many aspects, particularly nano-liquid chromatographic (LC) separation, mass spectrometric (MS)… (more)

▼ In the last decade, the field of proteomics has rapidly progressed with substantial advances in many aspects, particularly nano-liquid chromatographic (LC) separation, mass spectrometric (MS) instrumentation and bioinformatics tools. However, significant improvements are still needed to generate proteome-wide data for limited number of cells. By enhancing the sensitivity of the LC-MS analysis, identification of lower abundant proteins can be achieved. The work described in this thesis highlights some relevant advances that can help accelerate proteomics towards a high level of depth in regard to proteome coverage and how these achievements can find ample application in several research lines. First, recent advances in peptide separation by multidimensional liquid chromatography are reviewed. The most common LC-based techniques employed in proteomics are reversed-phase (RP), ion-exchange (IEX) and hydrophilic interaction liquid chromatography (HILIC). A detailed overview of these separations, as well their combination in multidimensional formats, are provided. Then, we introduce and evaluate the use of HILIC-based strategies for the separation of complex peptide mixtures. Two zwitterionic stationary phases, ZIC-HILIC and ZIC-cHILIC, differing in the spatial orientation of their charged groups on the chromatographic material, are fully characterized with respect to separation efficiency and a number of peptide physico-chemical properties affecting peptide retention. We extensively tested the performances of these HILIC materials in 2D-LC strategy in combination with RP for the analysis of low amounts (few micrograms) of human cell digests showing that HILIC can rival traditional multidimensional strategies employed in proteomics. Next, we apply this 2D strategy to the analysis of a limited number of FACS-sorted colon stem cells. With an optimized sample preparation and workflow, we enabled for the first time the in-depth global proteome analysis of 10,000 colon stem cells, obtaining a high proteome coverage. Moreover, we describe the feasibility of the dimethyl labeling method in combination with ZIC-cHILIC technology for quantitative proteomics. We address the potential issue of isotope effects perturbing the essential co-elution of differently labeled peptides under ZIC-cHILIC separation. The deuterium effect can be largely eliminated by choosing appropriate pH conditions, and an optimized approach at pH 3 is a suitable quantitative strategy. Furthermore, we combine the HILIC separation with a phosphopeptide enrichment approach based on Ti4+-IMAC to develop an efficient approach with the aim of maximizing the coverage of the cellular phosphoproteome. We design and systematically compare three strategies including: a sole Ti4+-IMAC enrichment; Ti4+-IMAC enrichment followed by HILIC fractionation; a pre-fractionation based on strong cation exchange, followed by Ti4+-IMAC enrichment and a further step of fractionation by HILIC. This work demonstrates the need to carry out extensive fractionations for deep mining of the… Advisors/Committee Members: Heck, Albert, Mohammed, Shabaz.

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APA (6^th Edition):

Di Palma, S. (2013). Optimizing hydrophilic interaction liquid chromatography for ultrasensitive proteome analysis. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/261028 ; URN:NBN:NL:UI:10-1874-261028 ; 1874/261028 ; urn:isbn:9789088915581 ; URN:NBN:NL:UI:10-1874-261028 ; https://dspace.library.uu.nl/handle/1874/261028

Chicago Manual of Style (16^th Edition):

Di Palma, S. “Optimizing hydrophilic interaction liquid chromatography for ultrasensitive proteome analysis.” 2013. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/261028 ; URN:NBN:NL:UI:10-1874-261028 ; 1874/261028 ; urn:isbn:9789088915581 ; URN:NBN:NL:UI:10-1874-261028 ; https://dspace.library.uu.nl/handle/1874/261028.

MLA Handbook (7^th Edition):

Di Palma, S. “Optimizing hydrophilic interaction liquid chromatography for ultrasensitive proteome analysis.” 2013. Web. 22 Jan 2021.

Vancouver:

Di Palma S. Optimizing hydrophilic interaction liquid chromatography for ultrasensitive proteome analysis. [Internet] [Doctoral dissertation]. University Utrecht; 2013. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/261028 ; URN:NBN:NL:UI:10-1874-261028 ; 1874/261028 ; urn:isbn:9789088915581 ; URN:NBN:NL:UI:10-1874-261028 ; https://dspace.library.uu.nl/handle/1874/261028.

Council of Science Editors:

Di Palma S. Optimizing hydrophilic interaction liquid chromatography for ultrasensitive proteome analysis. [Doctoral Dissertation]. University Utrecht; 2013. Available from: https://dspace.library.uu.nl/handle/1874/261028 ; URN:NBN:NL:UI:10-1874-261028 ; 1874/261028 ; urn:isbn:9789088915581 ; URN:NBN:NL:UI:10-1874-261028 ; https://dspace.library.uu.nl/handle/1874/261028

14. Gouw, J.W. Quantitative proteomics on the fly.

Degree: 2009, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/31857 ; URN:NBN:NL:UI:10-1874-31857 ; 1874/31857 ; urn:isbn:9789039349892 ; URN:NBN:NL:UI:10-1874-31857 ; https://dspace.library.uu.nl/handle/1874/31857

► The development of multicellular organisms is characterized by complex processes that progressively transform essentially a single cell into a creature with complicated structures and highly… (more)

▼ The development of multicellular organisms is characterized by complex processes that progressively transform essentially a single cell into a creature with complicated structures and highly specialized functions. The fruit fly Drosophila melanogaster provides an excellent model system to investigate these principles of life in great detail. Over the past few decades, Drosophila has been used to investigate and elucidate fundamental aspects that underlie the mechanisms described above. Since this research mainly involved large-scale analysis of genes, many principles are now understood at the gene level. However, it is nowadays clear that transcript abundances no not necessarily correlate with protein expression levels and since the latter determine the complexity of cells it is the ultimate goal to investigate and understand these processes directly at the protein level. The maturation of proteomics-based mass spectrometric techniques allows capturing the identity of many proteins in a single experiment. The primary goal of the work described in this thesis focused on gaining insights into the dynamics of early embryonic development of the fruit fly Drosophila melanogaster at the protein level by combining stable isotope labeling with high-accuracy mass spectrometry. We have developed a quantitative proteomic approach utilizing in vivo 15N-labeling of fruit flies with stable isotopes combined with extensive analysis by LC-MS/MS which has permitted the relative quantitation of thousands of proteins during early embryonic development. This provided insight into the production, stability and modification of individual proteins, while discrepancies between transcriptional profiles and protein dynamics indicated novel control mechanisms in genome activation during early fly development. Differential regulation of numerous proteins was observed and allowed the classification of these proteins into specialized classes. Although this approach shows its strength in identifying and quantitating proteins, some drawbacks include suboptimal labeling and lack of appropriate software for data processing. To overcome these issues, alternative objectives were explored in the work described in this thesis and aimed at developing methods to optimize, improve or facilitate the quantitative analysis of multiple proteins in a single experiment. Metabolic labeling with suboptimal (<98%) 15N-enrichments negatively affects protein identification and quantitation which was discovered by the systematic investigation of two independent 15N-labeled datasets. To overcome or compensate these shortcomings we have developed methods that can be applied to qualitative and quantitative 15N-labeled data. Although metabolic labeling produces most accurate quantitative data, it is not always feasible to incorporate an internal standard metabolically into an organism. So-called label-free techniques allow the quantitation of proteins in two or more samples without the need of using an internal standard. By directly comparing protein expression levels… Advisors/Committee Members: Heck, Albert, Krijgsveld, Jeroen.

Subjects/Keywords: Farmacie/Biofarmaceutische wetenschappen (FARM); Farmacie(FARM)

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APA (6^th Edition):

Gouw, J. W. (2009). Quantitative proteomics on the fly. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/31857 ; URN:NBN:NL:UI:10-1874-31857 ; 1874/31857 ; urn:isbn:9789039349892 ; URN:NBN:NL:UI:10-1874-31857 ; https://dspace.library.uu.nl/handle/1874/31857

Chicago Manual of Style (16^th Edition):

Gouw, J W. “Quantitative proteomics on the fly.” 2009. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/31857 ; URN:NBN:NL:UI:10-1874-31857 ; 1874/31857 ; urn:isbn:9789039349892 ; URN:NBN:NL:UI:10-1874-31857 ; https://dspace.library.uu.nl/handle/1874/31857.

MLA Handbook (7^th Edition):

Gouw, J W. “Quantitative proteomics on the fly.” 2009. Web. 22 Jan 2021.

Vancouver:

Gouw JW. Quantitative proteomics on the fly. [Internet] [Doctoral dissertation]. University Utrecht; 2009. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/31857 ; URN:NBN:NL:UI:10-1874-31857 ; 1874/31857 ; urn:isbn:9789039349892 ; URN:NBN:NL:UI:10-1874-31857 ; https://dspace.library.uu.nl/handle/1874/31857.

Council of Science Editors:

Gouw JW. Quantitative proteomics on the fly. [Doctoral Dissertation]. University Utrecht; 2009. Available from: https://dspace.library.uu.nl/handle/1874/31857 ; URN:NBN:NL:UI:10-1874-31857 ; 1874/31857 ; urn:isbn:9789039349892 ; URN:NBN:NL:UI:10-1874-31857 ; https://dspace.library.uu.nl/handle/1874/31857

15. Boersema, P.J. Advancing liquid chromatography- mass spectrometry based technologies for proteome research.

Degree: 2010, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/37547 ; URN:NBN:NL:UI:10-1874-37547 ; 1874/37547 ; urn:isbn:9789039352656 ; URN:NBN:NL:UI:10-1874-37547 ; https://dspace.library.uu.nl/handle/1874/37547

► In proteomics, high-tech nano-liquid chromatography (LC) and mass spectrometry (MS) instrumentation is used to routinely sequence proteins at a large scale. In this thesis, several… (more)

▼ In proteomics, high-tech nano-liquid chromatography (LC) and mass spectrometry (MS) instrumentation is used to routinely sequence proteins at a large scale. In this thesis, several technological developments are described to advance proteomics and their applicability is demonstrated in several different research lines. HILIC is an LC phase that exhibits some features that can be utilized in proteomics. The orthogonality of separation with reversed phase-LC and the more even dispersion of peptides over the LC run, makes HILIC an adequate first dimension for the fractionation of complex peptide mixtures. A specific variety of HILIC has been evaluated and further optimized for two dimensional-LC; zwitterionic HILIC (ZIC-HILIC). A mixed mode separation was observed for ZIC-HILIC consisting of both electrostatic and polar interactions between the peptides and stationary phase. Compared to SCX, less clustering of the typically ubiquitous +2 and +3 charged peptides was observed. Furthermore, the development of a triplex stable isotope dimethyl labeling approach is reported. By using different isotopomers of formaldehyde and cyanoborohydride, three different dimethyl labels can be generated in order to simultaneously analyze peptides from three different samples by LC-MS. The approach uses cheap and readily available chemicals and therefore large amounts of sample can be labeled. Furthermore, the labeling reaction goes to close to completion and virtually any sample can be labeled as the sample is performed post-lysis and -digestion. The metalloendopeptidase Lys-N has been investigated for MALDI-MS/MS proteomics applications. Lys-N produces peptides with an N-terminal Lys residue and therefore the basicity is concentrated at the N-terminus. Fragmentation in MALDI of peptides where this N-terminal Lys is the only basic group results in the generation of primarily N-terminal fragments. The CID spectra are straightforward and the sequence can be easily read off since often complete sequence ladders of b-ions are present. Phosphorylated peptides typically fragment differently compared to non-modified peptides. In CID for example, fragmentation of phosphorylated peptides can result in spectra that are dominated by a single peak that represents the neutral loss of the phosphate group. In chapter 6, this neutral loss effect and ways to accommodate challenges of MS based analysis of phosphopeptides are reviewed. The analysis of Tyr phosphorylation is rather challenging due to the low levels of Tyr phosphorylation. The enrichment efficiency of a phosphopeptide immuno-affinity purification was shown to be rather high and more than 1000 phospho-Tyr peptides could be identified after two separate LC-MS runs. Triplex stable isotope dimethyl labeling was combined with phospho-Tyr immunoprecipitation. Tyr phosphorylation was profiled after pervanadate and EGF stimulation, respectively. The quantitative immunoprecipitation method was also applied to study the role of FGF-2 stimulation in human embryonic stem cells (hESCs). FGF-2 is… Advisors/Committee Members: Heck, Albert, Mohammed, Shabaz.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Boersema, P. J. (2010). Advancing liquid chromatography- mass spectrometry based technologies for proteome research. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/37547 ; URN:NBN:NL:UI:10-1874-37547 ; 1874/37547 ; urn:isbn:9789039352656 ; URN:NBN:NL:UI:10-1874-37547 ; https://dspace.library.uu.nl/handle/1874/37547

Chicago Manual of Style (16^th Edition):

Boersema, P J. “Advancing liquid chromatography- mass spectrometry based technologies for proteome research.” 2010. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/37547 ; URN:NBN:NL:UI:10-1874-37547 ; 1874/37547 ; urn:isbn:9789039352656 ; URN:NBN:NL:UI:10-1874-37547 ; https://dspace.library.uu.nl/handle/1874/37547.

MLA Handbook (7^th Edition):

Boersema, P J. “Advancing liquid chromatography- mass spectrometry based technologies for proteome research.” 2010. Web. 22 Jan 2021.

Vancouver:

Boersema PJ. Advancing liquid chromatography- mass spectrometry based technologies for proteome research. [Internet] [Doctoral dissertation]. University Utrecht; 2010. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/37547 ; URN:NBN:NL:UI:10-1874-37547 ; 1874/37547 ; urn:isbn:9789039352656 ; URN:NBN:NL:UI:10-1874-37547 ; https://dspace.library.uu.nl/handle/1874/37547.

Council of Science Editors:

Boersema PJ. Advancing liquid chromatography- mass spectrometry based technologies for proteome research. [Doctoral Dissertation]. University Utrecht; 2010. Available from: https://dspace.library.uu.nl/handle/1874/37547 ; URN:NBN:NL:UI:10-1874-37547 ; 1874/37547 ; urn:isbn:9789039352656 ; URN:NBN:NL:UI:10-1874-37547 ; https://dspace.library.uu.nl/handle/1874/37547

16. Helbig, A.O. Probing yeast molecular systems biology by new proteomics strategies.

Degree: 2010, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/179133 ; URN:NBN:NL:UI:10-1874-179133 ; 1874/179133 ; urn:isbn:9789039353745 ; URN:NBN:NL:UI:10-1874-179133 ; https://dspace.library.uu.nl/handle/1874/179133

► The characterization of protein architecture in complex cellular model systems such asthe unicellular yeast Saccharomyces cerevisiae has become one of the major biological challenges of… (more)

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Helbig, A. O. (2010). Probing yeast molecular systems biology by new proteomics strategies. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/179133 ; URN:NBN:NL:UI:10-1874-179133 ; 1874/179133 ; urn:isbn:9789039353745 ; URN:NBN:NL:UI:10-1874-179133 ; https://dspace.library.uu.nl/handle/1874/179133

Chicago Manual of Style (16^th Edition):

Helbig, A O. “Probing yeast molecular systems biology by new proteomics strategies.” 2010. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/179133 ; URN:NBN:NL:UI:10-1874-179133 ; 1874/179133 ; urn:isbn:9789039353745 ; URN:NBN:NL:UI:10-1874-179133 ; https://dspace.library.uu.nl/handle/1874/179133.

MLA Handbook (7^th Edition):

Helbig, A O. “Probing yeast molecular systems biology by new proteomics strategies.” 2010. Web. 22 Jan 2021.

Vancouver:

Helbig AO. Probing yeast molecular systems biology by new proteomics strategies. [Internet] [Doctoral dissertation]. University Utrecht; 2010. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/179133 ; URN:NBN:NL:UI:10-1874-179133 ; 1874/179133 ; urn:isbn:9789039353745 ; URN:NBN:NL:UI:10-1874-179133 ; https://dspace.library.uu.nl/handle/1874/179133.

Council of Science Editors:

Helbig AO. Probing yeast molecular systems biology by new proteomics strategies. [Doctoral Dissertation]. University Utrecht; 2010. Available from: https://dspace.library.uu.nl/handle/1874/179133 ; URN:NBN:NL:UI:10-1874-179133 ; 1874/179133 ; urn:isbn:9789039353745 ; URN:NBN:NL:UI:10-1874-179133 ; https://dspace.library.uu.nl/handle/1874/179133

17. Gauci, S. Divide and conquer strategies for the in-depth analysis of proteomes.

Degree: 2010, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/179283 ; URN:NBN:NL:UI:10-1874-179283 ; 1874/179283 ; urn:isbn:9789039353882 ; URN:NBN:NL:UI:10-1874-179283 ; https://dspace.library.uu.nl/handle/1874/179283

► Global and in-depth protein characterisation is a key fundamental step in the unravelling of biological processes in all living organisms. However, the characterisation of the… (more)

▼ Global and in-depth protein characterisation is a key fundamental step in the unravelling of biological processes in all living organisms. However, the characterisation of the protein content (i.e. the proteome) of a cell, tissue or organism is an extremely challenging task due to the large number of proteins present, and their relative abundance spanning several orders of magnitude. Despite recent revolutionary developments in mass spectrometry (MS) based protein and peptide identification techniques, which have increased the speed and sensitivity for peptide sequencing, the reduction of the complexity of a proteome prior to MS analysis is currently still a prerequisite for in-depth proteome analysis. Therefore, numerous pre-fractionation techniques have been introduced for global proteomic profiling, which ideally result in an increase of the total number of protein identifications, evidently at the expense of an increase in analysis time. In this thesis, various protein and peptide pre-fractionation techniques have been explored, optimized and applied to the characterization of proteomes of several different origins ranging from cells to organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and human Hek293 cells. These protein and peptide separation techniques have been used orthogonally or separately, but always in combination with high mass-accuracy mass spectrometers to not only identify the peptides, but additionally also to identify post-translational modifications, primarily phosphorylation and N-terminal acetylation. A significant part of this thesis has focused on the use of orthogonal separation techniques for the comprehensive characterisation of the yeast nuclear proteome. In this study, phosphocellulose p11 chromatography was evaluated as a generic pre-fractionation technique as well as an enrichment technique for DNA binding proteins. The work in this thesis has revealed that each of the used pre-fractionation techniques has its own merits. Therefore, the combined use of multiple approaches is of fundamental importance, especially for the identification of low abundant proteins as well as to increase the confidence of the identification of proteins and/or their post-translational modifications. Advisors/Committee Members: Heck, Albert, Krijgsveld, Jeroen.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Gauci, S. (2010). Divide and conquer strategies for the in-depth analysis of proteomes. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/179283 ; URN:NBN:NL:UI:10-1874-179283 ; 1874/179283 ; urn:isbn:9789039353882 ; URN:NBN:NL:UI:10-1874-179283 ; https://dspace.library.uu.nl/handle/1874/179283

Chicago Manual of Style (16^th Edition):

Gauci, S. “Divide and conquer strategies for the in-depth analysis of proteomes.” 2010. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/179283 ; URN:NBN:NL:UI:10-1874-179283 ; 1874/179283 ; urn:isbn:9789039353882 ; URN:NBN:NL:UI:10-1874-179283 ; https://dspace.library.uu.nl/handle/1874/179283.

MLA Handbook (7^th Edition):

Gauci, S. “Divide and conquer strategies for the in-depth analysis of proteomes.” 2010. Web. 22 Jan 2021.

Vancouver:

Gauci S. Divide and conquer strategies for the in-depth analysis of proteomes. [Internet] [Doctoral dissertation]. University Utrecht; 2010. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/179283 ; URN:NBN:NL:UI:10-1874-179283 ; 1874/179283 ; urn:isbn:9789039353882 ; URN:NBN:NL:UI:10-1874-179283 ; https://dspace.library.uu.nl/handle/1874/179283.

Council of Science Editors:

Gauci S. Divide and conquer strategies for the in-depth analysis of proteomes. [Doctoral Dissertation]. University Utrecht; 2010. Available from: https://dspace.library.uu.nl/handle/1874/179283 ; URN:NBN:NL:UI:10-1874-179283 ; 1874/179283 ; urn:isbn:9789039353882 ; URN:NBN:NL:UI:10-1874-179283 ; https://dspace.library.uu.nl/handle/1874/179283

18. Aye, T.T. Application of chemical proteomics to biomarker discovery in cardiac research.

Degree: 2010, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/180078 ; URN:NBN:NL:UI:10-1874-180078 ; 1874/180078 ; urn:isbn:9789039354025 ; URN:NBN:NL:UI:10-1874-180078 ; https://dspace.library.uu.nl/handle/1874/180078

► This thesis is primarily focused on (i.) exploring chemical probes to increase sensitivity and specificity for the investigation of low abundant cardiac proteins applicable to… (more)

▼ This thesis is primarily focused on (i.) exploring chemical probes to increase sensitivity and specificity for the investigation of low abundant cardiac proteins applicable to both biology and biomarker discovery, and (ii.) exploiting different aspects of mass spectrometry-based proteomics for building a concentration-based cardiac proteome inventory. Chapter 1 is an overview of the applications of proteomics in cardiac diseases, including detailed descriptions of proteomics platforms used to analyze protein expression, function and quantity. It is also illustrated label-based and label-free mass-spectrometry quantitation methods to monitor changes in the proteome. The traditional biomarker discovery approach is described, including animal models and clinical studies. Chapter 2 reviews the specific contribution of mass spectrometry to the understanding of two particular cardiac signaling pathways that evolve around the second messenger cyclic nucleotides cAMP and cGMP. Two major downstream effectors of these cyclic nucleotides are Protein Kinase A and Protein Kinase G. Their relations to scaffold proteins that compartmentalize these kinases are discussed. An overview of mass spectrometry-based studies such as native mass spectrometry, H/D exchange and ion mobility mass spectrometry were described. In chapter 3, a chemical proteomics approach is combined with stable isotope labeling and mass spectrometry to study the specificity of different PKA isoforms for different AKAPs. Three different immobilized cAMP-analogues were used to enrich for PKA from several cell types and rat tissues. Stable isotope labeling was used to quantify the differential enrichment of the PKA-isoforms and thus their interacting AKAPs. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual-specificity with a potential preference for RI. For the first time, the specificity of AKAP14, AKAP2 and AKAP12 could be established. In Chapter 4, cAMP-based chemical proteomics is employed to identify potential changes in concentration and association of PKA in the dilated cardiomyopathy (DCM) affected human heart. Specific enrichment of PKA, PKG, several phosphodiesterases and many AKAPs from both healthy and DCM hearts were quantified in a label-free manner. We confirmed that PKA-R concentrations were lower in DCM affected tissue. Interestingly, the specific interactions of PKA with AKAPs were altered in the diseased heart under DCM conditions. These experiments, demonstrated the powerful combination of chemical proteomics with isotopic labeling as a potential application for cardiac biomarker discovery. Chapter 5 continues with the application of a multiplexed proteomics approach to generate a concentration-based human left ventricle proteome library. By using different separation methods/ proteases/ gas phase fragmentation methods, we identified 3,584 distinct proteins with high confidence which were quantitated using a sophisticated label-free spectral counting method to yield a comprehensive… Advisors/Committee Members: Heck, Albert, Scholten, Arjen.

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APA (6^th Edition):

Aye, T. T. (2010). Application of chemical proteomics to biomarker discovery in cardiac research. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/180078 ; URN:NBN:NL:UI:10-1874-180078 ; 1874/180078 ; urn:isbn:9789039354025 ; URN:NBN:NL:UI:10-1874-180078 ; https://dspace.library.uu.nl/handle/1874/180078

Chicago Manual of Style (16^th Edition):

Aye, T T. “Application of chemical proteomics to biomarker discovery in cardiac research.” 2010. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/180078 ; URN:NBN:NL:UI:10-1874-180078 ; 1874/180078 ; urn:isbn:9789039354025 ; URN:NBN:NL:UI:10-1874-180078 ; https://dspace.library.uu.nl/handle/1874/180078.

MLA Handbook (7^th Edition):

Aye, T T. “Application of chemical proteomics to biomarker discovery in cardiac research.” 2010. Web. 22 Jan 2021.

Vancouver:

Aye TT. Application of chemical proteomics to biomarker discovery in cardiac research. [Internet] [Doctoral dissertation]. University Utrecht; 2010. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/180078 ; URN:NBN:NL:UI:10-1874-180078 ; 1874/180078 ; urn:isbn:9789039354025 ; URN:NBN:NL:UI:10-1874-180078 ; https://dspace.library.uu.nl/handle/1874/180078.

Council of Science Editors:

Aye TT. Application of chemical proteomics to biomarker discovery in cardiac research. [Doctoral Dissertation]. University Utrecht; 2010. Available from: https://dspace.library.uu.nl/handle/1874/180078 ; URN:NBN:NL:UI:10-1874-180078 ; 1874/180078 ; urn:isbn:9789039354025 ; URN:NBN:NL:UI:10-1874-180078 ; https://dspace.library.uu.nl/handle/1874/180078

19. Ms. Taouatas, N. Lys-N: A versatile enzyme for proteomics.

Degree: 2010, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/193872 ; URN:NBN:NL:UI:10-1874-193872 ; 1874/193872 ; urn:isbn:9789039354889 ; URN:NBN:NL:UI:10-1874-193872 ; https://dspace.library.uu.nl/handle/1874/193872

► To overcome the difficulties of analyzing proteins in highly complex samples an improvement in proteomics strategies is needed. The combination of multiple proteases, peptide separation… (more)

▼ To overcome the difficulties of analyzing proteins in highly complex samples an improvement in proteomics strategies is needed. The combination of multiple proteases, peptide separation and fragmentation techniques may reduce sample complexity and improve the analysis of different sub-groups of peptides, including low abundant proteins and peptides. In this thesis, I introduce a relatively new protease in proteomics workflows and demonstrate the strength of combining its proteolytic peptides with multi-dimensional separation techniques and different fragmentation techniques, to decrease sample complexity and to improve sample identification. In chapter 2, we evaluate the fragmentation pattern observed for peptides generated by the metalloendopeptidase Lys-N using electron transfer dissociation (ETD). The enzyme Lys-N generates peptides with a lysine residue at the N-terminal. We show that ETD sequencing of BSA generated peptides with an N-terminal lysine, and no other basic residue in the sequence, result in spectra dominated by c-type fragment ions. To confirm the result, fragment ion statistics were increased by analyzing Lys-N generated peptides from a cell lysate. Additionally, we show that these doubly charged Lys-N peptides containing a single lysine at the N-terminal can be selectively enriched for by using low-pH strong cation exchange (SCX). In chapter 3, we further evaluate the SCX based fractionation of peptides generated from the metalloendopeptidase Lys-N. Here, we interestingly show that it is possible to obtain fractionation profiles where different subgroups of Lys-N generated peptides such as, acetylated N-terminal peptides, singly phosphorylated peptides, peptides with a single basic residue and peptides with multiple basic residues can be separated. We demonstrate that the combination of Lys-N digestion, low-pH SCX and reversed phase (RP) separation, with CID and ETD induced fragmentation, is a powerful approach for global proteome and phosphoproteome analysis. In chapter 4, the metalloendopeptidase was explored for its use in MALDI-MS/MS proteomics applications. Lys-N generated peptides from a BSA digest were analyzed by MALDI-MS/MS, which resulted in simple and straightforward CID spectra, containing complete b-ion series. Statistical analysis was again performed to confirm the results where a cell lysate was digested to obtain a higher number of doubly charged Lys-N peptides. Last, it was found that the simple straightforward MALDI CID spectra can be used to facilitate de novo sequencing. In chapter 5, the proteolytic performance of the metalloendopeptidase Lys-N was evaluated. As a model system BSA was used to validate the performance of Lys-N when using a number of classical proteomics sample handling conditions. We demonstrate that Lys-N has many interesting and useful characteristics as it was found to be highly thermo-stable and to have a high tolerance towards certain denaturing agents, such as urea and acetonitrile. Furthermore, it was found that by increasing the digestion temperature a… Advisors/Committee Members: Heck, Albert, Mohammed, Shabaz.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Ms. Taouatas, N. (2010). Lys-N: A versatile enzyme for proteomics. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/193872 ; URN:NBN:NL:UI:10-1874-193872 ; 1874/193872 ; urn:isbn:9789039354889 ; URN:NBN:NL:UI:10-1874-193872 ; https://dspace.library.uu.nl/handle/1874/193872

Chicago Manual of Style (16^th Edition):

Ms. Taouatas, N. “Lys-N: A versatile enzyme for proteomics.” 2010. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/193872 ; URN:NBN:NL:UI:10-1874-193872 ; 1874/193872 ; urn:isbn:9789039354889 ; URN:NBN:NL:UI:10-1874-193872 ; https://dspace.library.uu.nl/handle/1874/193872.

MLA Handbook (7^th Edition):

Ms. Taouatas, N. “Lys-N: A versatile enzyme for proteomics.” 2010. Web. 22 Jan 2021.

Vancouver:

Ms. Taouatas N. Lys-N: A versatile enzyme for proteomics. [Internet] [Doctoral dissertation]. University Utrecht; 2010. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/193872 ; URN:NBN:NL:UI:10-1874-193872 ; 1874/193872 ; urn:isbn:9789039354889 ; URN:NBN:NL:UI:10-1874-193872 ; https://dspace.library.uu.nl/handle/1874/193872.

Council of Science Editors:

Ms. Taouatas N. Lys-N: A versatile enzyme for proteomics. [Doctoral Dissertation]. University Utrecht; 2010. Available from: https://dspace.library.uu.nl/handle/1874/193872 ; URN:NBN:NL:UI:10-1874-193872 ; 1874/193872 ; urn:isbn:9789039354889 ; URN:NBN:NL:UI:10-1874-193872 ; https://dspace.library.uu.nl/handle/1874/193872

20. Hennrich, M.L. Expansion of the toolbox to decipher the (phospho)proteome.

Degree: 2012, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/235597 ; URN:NBN:NL:UI:10-1874-235597 ; 1874/235597 ; urn:isbn:9789039357583 ; URN:NBN:NL:UI:10-1874-235597 ; https://dspace.library.uu.nl/handle/1874/235597

► Nowadays, the method of choice to analyze proteins and peptides is mass spectrometry (MS). Still, several issues in the analysis of especially large pools of… (more)

▼ Nowadays, the method of choice to analyze proteins and peptides is mass spectrometry (MS). Still, several issues in the analysis of especially large pools of proteins like total cell lysates need to be solved to enable complete coverage of all proteins present in such a sample. In this thesis several novel methods for a better identification of peptides are described. After an introduction about the basics of mass spectrometry based proteomics, the most common liquid chromatography based separation techniques in proteomics are reviewed and a detailed overview over the principles of these separation techniques and their combination in multidimensional separations is given. Today, most mass spectrometric data of peptide analysis are interpreted by comparison with in silico generated spectra derived from protein or genomic databases. If the sequence information of the protein/peptide of interest is not available in the database, de novo sequencing is the method of choice. In this thesis a novel method for de novo sequencing of peptides is described. The method is based on the digestion of the proteins of interest with Lys-N metalloendoprotease, which enables specific labeling of solely the N-terminus. Differential isotopic dimethyl labeling of the peptides combined with simultaneous fragmentation of the isotopologues results in specific isotopic patterns of N-terminal fragments, which enables facile de novo sequencing. By this methodology we were able to partially sequence a previously unknown protein from avocado fruit. Another methodology for de novo sequencing is based on the analysis of doubly charged Lys-N peptides with ETD. In this thesis, we explored the fragmentation behavior upon ETD of doubly charged tryptic and Lys-N peptides derivatized with some commonly applied labels. Increased sequence coverage and simplification of spectra for some labels and suppression of fragmentation for nicotinylated peptides are described in detail. Some pools of peptides like phosphorylated peptides are often present in much lower concentration as the bulk of regular peptides. Therefore, enrichment strategies like strong cation exchange (SCX) chromatography have been developed. In this thesis, we applied weak anion exchange to further decrease the complexity of the enriched phosphopeptide fractions from SCX. As a result, we were able to identify more than 10,000 phosphopeptides out of a single SCX fraction. Unfortunately, phosphorylated peptides that contain multiple basic residues elute with the bulk of regular peptides in SCX. Thus, further enrichment is needed. We developed a facile method to enrich for these pools of phosphorylated peptides. Individual fractions from the first SCX separation at a pH of 3 are further separated with SCX at a pH of 1. Due to the change of net charge of the phosphopeptides but not of regular peptides, separation and enrichment of phosphorylated peptides in this tandem SCX approach is possible. By this method we were able to identify more than 10,000 phosphopeptides out of only 500 µg of protein.… Advisors/Committee Members: Heck, Albert, Mohammed, Shabaz.

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APA (6^th Edition):

Hennrich, M. L. (2012). Expansion of the toolbox to decipher the (phospho)proteome. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/235597 ; URN:NBN:NL:UI:10-1874-235597 ; 1874/235597 ; urn:isbn:9789039357583 ; URN:NBN:NL:UI:10-1874-235597 ; https://dspace.library.uu.nl/handle/1874/235597

Chicago Manual of Style (16^th Edition):

Hennrich, M L. “Expansion of the toolbox to decipher the (phospho)proteome.” 2012. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/235597 ; URN:NBN:NL:UI:10-1874-235597 ; 1874/235597 ; urn:isbn:9789039357583 ; URN:NBN:NL:UI:10-1874-235597 ; https://dspace.library.uu.nl/handle/1874/235597.

MLA Handbook (7^th Edition):

Hennrich, M L. “Expansion of the toolbox to decipher the (phospho)proteome.” 2012. Web. 22 Jan 2021.

Vancouver:

Hennrich ML. Expansion of the toolbox to decipher the (phospho)proteome. [Internet] [Doctoral dissertation]. University Utrecht; 2012. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/235597 ; URN:NBN:NL:UI:10-1874-235597 ; 1874/235597 ; urn:isbn:9789039357583 ; URN:NBN:NL:UI:10-1874-235597 ; https://dspace.library.uu.nl/handle/1874/235597.

Council of Science Editors:

Hennrich ML. Expansion of the toolbox to decipher the (phospho)proteome. [Doctoral Dissertation]. University Utrecht; 2012. Available from: https://dspace.library.uu.nl/handle/1874/235597 ; URN:NBN:NL:UI:10-1874-235597 ; 1874/235597 ; urn:isbn:9789039357583 ; URN:NBN:NL:UI:10-1874-235597 ; https://dspace.library.uu.nl/handle/1874/235597

21. Mithoe, S.C. Modulation of MAPK signaling in plant immunity and development.

Degree: 2013, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/276685 ; URN:NBN:NL:UI:10-1874-276685 ; 1874/276685 ; urn:isbn:9789088916564 ; URN:NBN:NL:UI:10-1874-276685 ; https://dspace.library.uu.nl/handle/1874/276685

► Plants and animal cells use intricate signaling pathways to respond to a diverse array of stimuli. These stimuli include signals from the environment, such as… (more)

▼ Plants and animal cells use intricate signaling pathways to respond to a diverse array of stimuli. These stimuli include signals from the environment, such as biotic and abiotic stress signals, as well as cell-to-cell signaling required for pattern formation during development. The transduction of the signal often relies on protein phosphorylation, which in eukaryotic cells occurs mainly on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. In animal systems Tyr phosphorylation plays a prominent role in signaling, however the relative contribution of Tyr phosphorylation to plant signal transduction has remained an open question. We implemented an approach to selectively measure and quantify Tyr phosphorylation in plant cells, which can also be applied to whole plants. We reproducibly quantified 23 pTyr peptides in two inversely labeled biological replicates, identifying 11 differentially phosphorylated proteins. These include a set of 3 well-characterized flagellin responsive mitogen-activated protein kinases (MAPKs) and 4 novel MAPKs. In plant signal transduction MAPKs are important in transducing signals from the upstream receptor to the downstream targets and they are pivotal signaling modules in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). In Arabidopsis thaliana (Arabidopsis), the MEK4/5-MPK3/6 module is an important part of a major MAPK cascade in immune responses. We report the functional analysis of a Arabidopsis MAPK kinase kinase (MAPKKK) as a negative regulator of PTI signaling and basal immunity. We show that this function requires phosphorylation of this protein on specific Ser residues. Based on genetic evidence we observed, that this MAPKKK is also involved in root meristem maintenance and meristem cell proliferation, which are required for root growth. Our results show that through phosphorylation of this MAPKKK, signaling pathways controlling growth and immune response are antagonistically regulated. Advisors/Committee Members: Pieterse, Corné, Heck, Albert.

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APA (6^th Edition):

Mithoe, S. C. (2013). Modulation of MAPK signaling in plant immunity and development. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/276685 ; URN:NBN:NL:UI:10-1874-276685 ; 1874/276685 ; urn:isbn:9789088916564 ; URN:NBN:NL:UI:10-1874-276685 ; https://dspace.library.uu.nl/handle/1874/276685

Chicago Manual of Style (16^th Edition):

Mithoe, S C. “Modulation of MAPK signaling in plant immunity and development.” 2013. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/276685 ; URN:NBN:NL:UI:10-1874-276685 ; 1874/276685 ; urn:isbn:9789088916564 ; URN:NBN:NL:UI:10-1874-276685 ; https://dspace.library.uu.nl/handle/1874/276685.

MLA Handbook (7^th Edition):

Mithoe, S C. “Modulation of MAPK signaling in plant immunity and development.” 2013. Web. 22 Jan 2021.

Vancouver:

Mithoe SC. Modulation of MAPK signaling in plant immunity and development. [Internet] [Doctoral dissertation]. University Utrecht; 2013. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/276685 ; URN:NBN:NL:UI:10-1874-276685 ; 1874/276685 ; urn:isbn:9789088916564 ; URN:NBN:NL:UI:10-1874-276685 ; https://dspace.library.uu.nl/handle/1874/276685.

Council of Science Editors:

Mithoe SC. Modulation of MAPK signaling in plant immunity and development. [Doctoral Dissertation]. University Utrecht; 2013. Available from: https://dspace.library.uu.nl/handle/1874/276685 ; URN:NBN:NL:UI:10-1874-276685 ; 1874/276685 ; urn:isbn:9789088916564 ; URN:NBN:NL:UI:10-1874-276685 ; https://dspace.library.uu.nl/handle/1874/276685

22. Frese, C. Development and Application of Novel Electron Transfer Dissociation-based Technologies for Proteomics.

Degree: 2013, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/278011 ; URN:NBN:NL:UI:10-1874-278011 ; 1874/278011 ; urn:isbn:9789088916618 ; URN:NBN:NL:UI:10-1874-278011 ; https://dspace.library.uu.nl/handle/1874/278011

► Proteins are key actors in all cellular processes and pathways and almost all diseases are linked to perturbations of proteins, their modification state or interaction… (more)

▼ Proteins are key actors in all cellular processes and pathways and almost all diseases are linked to perturbations of proteins, their modification state or interaction networks. Mass spectrometry-based proteomics has matured to a high-throughput quantitative technology, aiming to provide sensitive, accurate and complete information on protein abundance, interactions and networks. In the current workflow in MS-based proteomics, proteins are cleaved into peptides by proteolytic digestion followed by MS analysis. Peptide sequencing is key to any MS-based proteomic experiment and provides the foundation for structural analysis. Collision induced dissociation (CID) is the most established dissociation technique and is nowadays routinely used for peptide sequencing. Over the last years, electron-driven approaches such as electron transfer dissociation (ETD) have evolved as valuable alternative fragmentation techniques. In this thesis the development of novel tandem mass spectrometry technologies based on electron-transfer dissociation is described. First, a new method that combines CID and ETD with either ion trap or Orbitrap detection in a data-dependent decision tree approach is introduced. This method enables selection of the most appropriate combination of fragmentation technique and mass analyzer ‘on-the-fly’, in turn leading to more confident peptide identification. Next, a novel fragmentation method combining higher-energy collision dissociation (HCD) and ETD in a single MS/MS event is described. This method, coined EThcD, provides extensive peptide backbone fragmentation and substantially increases the overall peptide sequence coverage. Furthermore, the higher quality in EThcD spectra facilitates the analysis of post-translational modifications, e.g. phosphorylation or glycosylation. Localization of post-translational modifications is challenging because it requires observation of site-determining fragment ions. This is exemplified for the analysis of phosphorylated peptides, which revealed superior phosphorylation site assignment using EThcD compared to current fragmentation methods. Another part of this thesis describes a novel instrumental setup to perform ETD reactions in the HCD collision cell on an Orbitrap Velos instrument by applying a static DC gradient axially to the rods. This gradient enables simultaneous three dimensional, charge sign independent, trapping of cations and anions, initiating electron transfer reactions in the center of the HCD cell where oppositely charged ions clouds overlap. The data shows that performing ETD in the HCD cell provides similar fragmentation as ion trap-ETD but will require further optimization to match performance of ion trap-ETD. The benefits of ETD are further highlighted in a study of endogenous neuropeptides derived from rat brain. Neuropeptides are key players in food intake regulation. Here, we analyze brain extract from two different brain areas. Based on the results from chapter 2, we utilized HCD and ETD fragmentation to increase the overall peptide… Advisors/Committee Members: Heck, Albert, Altelaar, Maarten.

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APA (6^th Edition):

Frese, C. (2013). Development and Application of Novel Electron Transfer Dissociation-based Technologies for Proteomics. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/278011 ; URN:NBN:NL:UI:10-1874-278011 ; 1874/278011 ; urn:isbn:9789088916618 ; URN:NBN:NL:UI:10-1874-278011 ; https://dspace.library.uu.nl/handle/1874/278011

Chicago Manual of Style (16^th Edition):

Frese, C. “Development and Application of Novel Electron Transfer Dissociation-based Technologies for Proteomics.” 2013. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/278011 ; URN:NBN:NL:UI:10-1874-278011 ; 1874/278011 ; urn:isbn:9789088916618 ; URN:NBN:NL:UI:10-1874-278011 ; https://dspace.library.uu.nl/handle/1874/278011.

MLA Handbook (7^th Edition):

Frese, C. “Development and Application of Novel Electron Transfer Dissociation-based Technologies for Proteomics.” 2013. Web. 22 Jan 2021.

Vancouver:

Frese C. Development and Application of Novel Electron Transfer Dissociation-based Technologies for Proteomics. [Internet] [Doctoral dissertation]. University Utrecht; 2013. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/278011 ; URN:NBN:NL:UI:10-1874-278011 ; 1874/278011 ; urn:isbn:9789088916618 ; URN:NBN:NL:UI:10-1874-278011 ; https://dspace.library.uu.nl/handle/1874/278011.

Council of Science Editors:

Frese C. Development and Application of Novel Electron Transfer Dissociation-based Technologies for Proteomics. [Doctoral Dissertation]. University Utrecht; 2013. Available from: https://dspace.library.uu.nl/handle/1874/278011 ; URN:NBN:NL:UI:10-1874-278011 ; 1874/278011 ; urn:isbn:9789088916618 ; URN:NBN:NL:UI:10-1874-278011 ; https://dspace.library.uu.nl/handle/1874/278011

23. de Graaf, E.L. Novel (pospho-)proteomics approaches to study aging and senescence.

Degree: 2014, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/289092 ; URN:NBN:NL:UI:10-1874-289092 ; 1874/289092 ; urn:isbn:9789088918049 ; URN:NBN:NL:UI:10-1874-289092 ; https://dspace.library.uu.nl/handle/1874/289092

► This thesis describes the application of proteomics technologies to further increase the knowledge about aging and cancer suppression mechanisms. The core tool in all performed… (more)

▼ This thesis describes the application of proteomics technologies to further increase the knowledge about aging and cancer suppression mechanisms. The core tool in all performed experiments is mass spectrometry based proteomics. In addition to the application of these techniques, newly developed proteomics techniques are being introduced that help to increase the analytical power of proteomics experiments. In chapter one, a general introduction is given into proteomics and mass spectrometry-based proteomics workflows. The type of research question strongly determines the type of experimental workflow and equipment needed. Therefore, there is no standard one method fits all approach resulting in highly dynamic experimental workflow with a myriad of different techniques/approaches. In chapter two, the influence of DNA-damage on tissue aging was studied for a specific part of the mouse brain over time. Quantitative shotgun proteomics discovery experiments were performed in order to find biomarkers and mechanisms of aging in the cerebellum. After the identification of several interesting differentially regulated proteins associated with age and neuron-deterioration by proteomics, a more targeted validation was performed using immunohistochemistry. By using this complementary method, changes over time as well as changes in protein localization could be monitored for a set of differentially regulated proteins. Major observations of this study include a DNA-damage induced down regulation of a tightly interconnected synaptic signaling network associated with a morphological transformation of Purkinje cells and a new molecular link between DNA-damage and motoric diseases such as spinocerebellar ataxia. Chapter three describes a technical method development to optimize the transition from a discovery orientated proteomics approach to a targeted proteomics experiment. The work demonstrates that CID spectra acquired on SRM instruments are more similar to spectra acquired in the HCD cell than those acquired in the ion trap of hybrid LTQ-Orbitrap instruments. Concomitantly, SRM assays generated using HCD spectra showed a higher sensitivity when compared to ion trap spectra-generated SRM assays. Therefore, when planning a targeted MS experiment, choosing for HCD fragmentation in the discovery phase can help facilitate SRM assay development later on. In chapter four, the phosphopeptide enrichment robustness of a new Ti4+-IMAC method was assessed. First it was established that Ti4+-IMAC enrichment resulted in a highly reproducible quantification of phosphorylation sites in HeLa cells. Subsequently, this strategy was applied to monitor the phosphoproteome of Jurkat T-cells upon Prostaglandin E2 stimulation over the timespan of 60 minutes. It was demonstrated that using this enrichment strategy and label-free quantification a comprehensive temporal phosphoproteome of Jurkat T-cells could be constructed, indicating differential regulation of different kinases over time. The proved straight-forward yet comprehensive phosphoproteomics… Advisors/Committee Members: Heck, Albert, Altelaar, Maarten.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

de Graaf, E. L. (2014). Novel (pospho-)proteomics approaches to study aging and senescence. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/289092 ; URN:NBN:NL:UI:10-1874-289092 ; 1874/289092 ; urn:isbn:9789088918049 ; URN:NBN:NL:UI:10-1874-289092 ; https://dspace.library.uu.nl/handle/1874/289092

Chicago Manual of Style (16^th Edition):

de Graaf, E L. “Novel (pospho-)proteomics approaches to study aging and senescence.” 2014. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/289092 ; URN:NBN:NL:UI:10-1874-289092 ; 1874/289092 ; urn:isbn:9789088918049 ; URN:NBN:NL:UI:10-1874-289092 ; https://dspace.library.uu.nl/handle/1874/289092.

MLA Handbook (7^th Edition):

de Graaf, E L. “Novel (pospho-)proteomics approaches to study aging and senescence.” 2014. Web. 22 Jan 2021.

Vancouver:

de Graaf EL. Novel (pospho-)proteomics approaches to study aging and senescence. [Internet] [Doctoral dissertation]. University Utrecht; 2014. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/289092 ; URN:NBN:NL:UI:10-1874-289092 ; 1874/289092 ; urn:isbn:9789088918049 ; URN:NBN:NL:UI:10-1874-289092 ; https://dspace.library.uu.nl/handle/1874/289092.

Council of Science Editors:

de Graaf EL. Novel (pospho-)proteomics approaches to study aging and senescence. [Doctoral Dissertation]. University Utrecht; 2014. Available from: https://dspace.library.uu.nl/handle/1874/289092 ; URN:NBN:NL:UI:10-1874-289092 ; 1874/289092 ; urn:isbn:9789088918049 ; URN:NBN:NL:UI:10-1874-289092 ; https://dspace.library.uu.nl/handle/1874/289092

24. Burgers, P.P. Identification and Structural Characterization of Novel A-kinase Anchoring Proteins.

Degree: 2014, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; 1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; https://dspace.library.uu.nl/handle/1874/294437

► The work described in this thesis initially focusses on the discovery of a novel PKA-RI specific AKAP: small membrane AKAP (smAKAP). Afterwards we centre on… (more)

▼ The work described in this thesis initially focusses on the discovery of a novel PKA-RI specific AKAP: small membrane AKAP (smAKAP). Afterwards we centre on the structural interaction between smAKAP and PKA-RI to reveal the first PKA-RI specific AKAP bound to PKA-RI crystal structure. Interestingly, a novel self-inhibition mechanism was discovered which allows PKA to block its binding to AKAPs under certain restrictions. In order to fully understand the specific limitations associated with binding a study centering on the PKA-RI and PKA-RII interactions with AKAPs was performed. In Chapter 2, recent literature on the structural interface between PKA-RI/RII and AKAPs is reviewed. Most of these structural studies involve either X-ray crystallography, three-dimensional NMR, binding affinity assays and various other biochemical methods. In Chapter 3, the discovery and initial characterization of a novel AKAP termed smAKAP is described. Via binding affinity assays and imaging techniques it is shown that smAKAP is PKA-RI specific. The intracellular location of smAKAP at the plasma membrane is shown by means of fluorescence imaging and advanced electron microscopy. In Chapter 4, structural techniques such as hydrogen/deuterium exchange and X-ray crystallography are applied to probe the interaction interface between smAKAP and PKA-RI. Additionally, via a phosphoproteomics study it was shown that in the middle of the A-kinase binding domain of smAKAP there is a putative PKA phosphorylation site. Upon phosphorylation of this site, PKA cannot bind to smAKAP anymore. A mechanistic model on how this disruption occurs is presented. In Chapter 5, a novel bioinformatic tool, THAHIT (THe AKAP/amphipathic Helix Identification Tool), is able to predict PKA-RIα and/or PKA-RIIα binding domains. This software package is based on currently known and well-established PKA-RIα and PKA-RIIα binding motifs. After applying it on all known AKAPs, numerous new PKA-RIα and PKA-RIIα binding domains in these AKAPs were found and/or narrowed down. Several of these were confirmed via conservation (BlastP), in silico docking studies using HADDOCK and in vitro binding studies using fluorescence anisotropy. In addition, several cAMP pull-downs were investigated for potential novel AKAPs using THAHIT. Here we propose a novel very large AKAP: vlAKAP. Advisors/Committee Members: Heck, Albert, Scholten, Arjen.

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APA (6^th Edition):

Burgers, P. P. (2014). Identification and Structural Characterization of Novel A-kinase Anchoring Proteins. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; 1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; https://dspace.library.uu.nl/handle/1874/294437

Chicago Manual of Style (16^th Edition):

Burgers, P P. “Identification and Structural Characterization of Novel A-kinase Anchoring Proteins.” 2014. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; 1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; https://dspace.library.uu.nl/handle/1874/294437.

MLA Handbook (7^th Edition):

Burgers, P P. “Identification and Structural Characterization of Novel A-kinase Anchoring Proteins.” 2014. Web. 22 Jan 2021.

Vancouver:

Burgers PP. Identification and Structural Characterization of Novel A-kinase Anchoring Proteins. [Internet] [Doctoral dissertation]. University Utrecht; 2014. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; 1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; https://dspace.library.uu.nl/handle/1874/294437.

Council of Science Editors:

Burgers PP. Identification and Structural Characterization of Novel A-kinase Anchoring Proteins. [Doctoral Dissertation]. University Utrecht; 2014. Available from: https://dspace.library.uu.nl/handle/1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; 1874/294437 ; URN:NBN:NL:UI:10-1874-294437 ; https://dspace.library.uu.nl/handle/1874/294437

25. Leite Guerreiro, A.C. A sneak preview of the daily life of S. elongatus by MS-based proteomics.

Degree: 2016, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/325246 ; URN:NBN:NL:UI:10-1874-325246 ; 1874/325246 ; urn:isbn:9789462954281 ; URN:NBN:NL:UI:10-1874-325246 ; https://dspace.library.uu.nl/handle/1874/325246

► Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi and several bacteria.… (more)

▼ Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi and several bacteria. The central mechanism behind these ‘pacemakers’ and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, named KaiA, KaiB and KaiC. This central clock system has been extensively studied functionally, structurally and can be reconstituted in vitro. These characteristics together with the relatively small genome (2.7 Mbp) of S. elongatus, make it an ideal model system for the study of circadian rhythms. To provide new insights for the molecular mechanisms of the S. elongatus circadian clock in particular, different quantitative proteomics and phosphoproteomics methods were used. First, high-resolution Mass Spectrometry (MS) was combined with the TMT 6-plex labeling technique to probe cyclic protein abundance variations across the S. elongatus proteome. This 48-hour time-series study revealed that, in contrast to abundant occurrences of cyclic and even circadian rhythms at the transcript level (30-60%), at the protein level these variations are much less pronounced, with only 5% of the quantified proteome showing significant cyclic variations. Next, the focus moved towards the analysis of protein interactions and complexes. A novel approach, combining native size exclusion chromatography and MS, was used to uncover differences in protein assemblies between light and dark states. Several proteins involved in higher order complexes, which didn’t show diurnal adaptation in protein abundance in the previous study, were found to have association dynamics. Some examples are the photosynthetic and ribosomal proteins, which showed differences in abundances as well as variable assemblies. Finally, the combination of MS, Ti4+-IMAC phosphopeptide enrichment and TMT 6-plex labeling was explored to uncover clues for regulation of molecular rhythms by protein phosphorylation. This first look at the phosphoproteome revealed new phosphorylation sites on proteins associated with diverse molecular pathways. Advisors/Committee Members: Heck, Albert, Altelaar, Maarten.

Subjects/Keywords: Mass spectrometry; Proteomics; Cyanobacteria; Circadian rhythm; Daily rhythms; Protein-protein interactions; Phosphoproteome

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APA (6^th Edition):

Leite Guerreiro, A. C. (2016). A sneak preview of the daily life of S. elongatus by MS-based proteomics. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/325246 ; URN:NBN:NL:UI:10-1874-325246 ; 1874/325246 ; urn:isbn:9789462954281 ; URN:NBN:NL:UI:10-1874-325246 ; https://dspace.library.uu.nl/handle/1874/325246

Chicago Manual of Style (16^th Edition):

Leite Guerreiro, A C. “A sneak preview of the daily life of S. elongatus by MS-based proteomics.” 2016. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/325246 ; URN:NBN:NL:UI:10-1874-325246 ; 1874/325246 ; urn:isbn:9789462954281 ; URN:NBN:NL:UI:10-1874-325246 ; https://dspace.library.uu.nl/handle/1874/325246.

MLA Handbook (7^th Edition):

Leite Guerreiro, A C. “A sneak preview of the daily life of S. elongatus by MS-based proteomics.” 2016. Web. 22 Jan 2021.

Vancouver:

Leite Guerreiro AC. A sneak preview of the daily life of S. elongatus by MS-based proteomics. [Internet] [Doctoral dissertation]. University Utrecht; 2016. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/325246 ; URN:NBN:NL:UI:10-1874-325246 ; 1874/325246 ; urn:isbn:9789462954281 ; URN:NBN:NL:UI:10-1874-325246 ; https://dspace.library.uu.nl/handle/1874/325246.

Council of Science Editors:

Leite Guerreiro AC. A sneak preview of the daily life of S. elongatus by MS-based proteomics. [Doctoral Dissertation]. University Utrecht; 2016. Available from: https://dspace.library.uu.nl/handle/1874/325246 ; URN:NBN:NL:UI:10-1874-325246 ; 1874/325246 ; urn:isbn:9789462954281 ; URN:NBN:NL:UI:10-1874-325246 ; https://dspace.library.uu.nl/handle/1874/325246

26. Marino, F. Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome.

Degree: 2016, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; 1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; https://dspace.library.uu.nl/handle/1874/338044

► The work, described in this thesis, has been aimed at the improvement of proteomic workflows; from the sample preparation, to the optimization of state-of-art chromatographical… (more)

▼ The work, described in this thesis, has been aimed at the improvement of proteomic workflows; from the sample preparation, to the optimization of state-of-art chromatographical separations and alternative MS fragmentation techniques. An important focus of this work is represented by the analysis of peptides harboring various post translational modifications (PTMs) such as glycosylation, phosphorylation, methylation and di-methylation. PTM modified peptides often require customized approaches in order to improve their detection. Furthermore advancements in LC-MS/MS approaches can enhance the detection of endogenous peptides eluted from HLA class I and II molecules, which present exotic characteristics when compared to ‘standard’ tryptic peptides obtained in proteomic workflows. First we discuss the development and implementation of an online 2D-dimensional SCX/RP UHPLC system coupled to a fast sequencing and high resolution MS. In this work we asked the question whether the fast and sensitive 2D LC-MS/MS workflow could compete with a 1D, in terms of proteome coverage, analysis time and sample usage. Next we explore a method based on several stages of state-of-art chromatographical techniques, high resolution MS and alternative fragmentation to define kinase consensus motifs with high accuracy which can be implemented for any kinase. We further demonstrate that recent developments in MS-based sequencing technology can expand the detectable peptide repertoire revealing unique features in the antigen presentation machinery.By analyzing a vast repertoires of HLA class I eluted peptides, we found that they can harbor, little explored PTMs such as arginine methylation and di-methylation. Interestingly, we also found evidences of unusual glycosylated HLA class I-bound peptides. The observed O-linked glycans are extended O-GlcNAc rather than GalNAc-initiated O-glycans. In fact O-GlcNAc on an HLA peptide may present a special case because of the HLA class I pathway traffics through the ER and Golgi on its way to the cell membrane, and can hence be exposed to glycosyltransferases that further modify the initiator O-GlcNAc group. In another work on PTMs harboring HLA class I peptides, we charted the phosphopeptides bound to four distinct HLA-B molecules and observed that they shared a preference for peptides phosphorylated at position 4. We investigated the structural basis for this observation concluding that the observed phosphorylation motif may be common to most HLA-B molecules.Alterations in PTMs are a recognized hallmark of diseases, therefore these kind of PTMs can potentially provide a unique source of HLA class I-presented peptide antigens that could uniquely stimulate the immune response. Moreover, the knowledge added by our works provides a base for the improved prediction and identification of PTMs neo-antigens, as potentially used for cancer immunotherapy. Lastly, we evaluate a mass MS-based analysis of HLA-DR-associated self-peptides. The complementary peptide fragmentation techniques ETD, EThcD and HCD were… Advisors/Committee Members: Heck, Albert, Mohammed, Shabaz.

Subjects/Keywords: Alternative fragmentations; HLA class I; HLA class II; phosphorylation; methylation; glycosylation

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APA (6^th Edition):

Marino, F. (2016). Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; 1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; https://dspace.library.uu.nl/handle/1874/338044

Chicago Manual of Style (16^th Edition):

Marino, F. “Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome.” 2016. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; 1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; https://dspace.library.uu.nl/handle/1874/338044.

MLA Handbook (7^th Edition):

Marino, F. “Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome.” 2016. Web. 22 Jan 2021.

Vancouver:

Marino F. Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome. [Internet] [Doctoral dissertation]. University Utrecht; 2016. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; 1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; https://dspace.library.uu.nl/handle/1874/338044.

Council of Science Editors:

Marino F. Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome. [Doctoral Dissertation]. University Utrecht; 2016. Available from: https://dspace.library.uu.nl/handle/1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; 1874/338044 ; URN:NBN:NL:UI:10-1874-338044 ; https://dspace.library.uu.nl/handle/1874/338044

27. Cabukusta, Birol. Molecular Dissection of a Candidate Ceramide Sensor.

Degree: 2016, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/341578 ; URN:NBN:NL:UI:10-1874-341578 ; 1874/341578 ; urn:isbn:9789462955493 ; URN:NBN:NL:UI:10-1874-341578 ; https://dspace.library.uu.nl/handle/1874/341578

► Sphingomyelin is an essential component of cellular membranes that contribute to the barrier function of the plasma membrane, signaling and molecular sorting. Ceramides are precursors… (more)

▼ Sphingomyelin is an essential component of cellular membranes that contribute to the barrier function of the plasma membrane, signaling and molecular sorting. Ceramides are precursors of sphingomyelins and they are produced de novo in the ER. Ceramides are also associated with growth arrest, senescence and apoptosis. Thus, cells must control their ceramides to keep the balance between proliferation and growth arrest/apoptosis. The bulk of newly synthesized ceramides is converted to sphingomyelin in the Golgi by sphingomyelin synthases (SMS). Mammalian cells contain two SMS isoforms, SMS1 in the Golgi and SMS2 at the plasma membrane. A closely related third enzyme, sphingomyelin synthase-related protein (SMSr) is not a conventional SM synthase but synthesizes ceramide-phosphoethanolamine. SMSr orthologs are found in all members of the animal kingdom, even though some do not produce SM. We recently suggested that SMSr is a candidate ceramide sensor whose primary role is to monitor ER ceramide levels to protect cells against the potential risk of apoptosis during sphingolipid biosynthesis. The present thesis aims to investigate the molecular mechanisms by which SMSr regulates ceramide homeostasis. We found that SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Alzheimer’s Huntington’s disease. In cells treated with staurosporine or FasL, SMSr undergoes caspase-6-dependent cleavage at a conserved aspartate located between its N-terminal SAM domain and the first membrane span. Next, we uncovered a striking structural and functional similarity between SMSr-SAM and the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. We demonstrated that SMSr-SAM drives self-assembly of the enzyme into ER-resident trimers and hexamers. Mutations that destabilize SMSr oligomers caused a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and calcium homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Moreover, we report on the successful application of single-molecule photobleaching as approach to monitor homo-typic oligomerization of SMSr in its native cellular environment. By imaging GFP-tagged SMSr proteins as single-fluorescent spots in the ER of intact cells, we were able to trace photobleaching of protein complexes to monitor their oligomeric states. In agreement with our biochemical data, this analysis shows that the SAM domain of SMSr drives self-assembly of the enzyme in the ER and that curcumin promotes SMSr oligomerization. Our study documents, for the first time, that single-molecule photobleaching can be used to resolve changes in the oligomeric state of an ER-resident membrane protein, hence establishing a valuable complementary method to unravel the mechanism by which SMSr mediates ceramide homeostasis in the ER. Collectively, our findings in this thesis have resulted in a better understanding and a… Advisors/Committee Members: Holthuis, Joost, Heck, Albert.

Subjects/Keywords: Lipids; Ceramide; Sphingomyelin; Endoplasmic Reticulum; Apoptosis; Homo-oligomerization; Single-molecule microscopy; SMSr; SAMD8; curcumin

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APA (6^th Edition):

Cabukusta, B. (2016). Molecular Dissection of a Candidate Ceramide Sensor. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/341578 ; URN:NBN:NL:UI:10-1874-341578 ; 1874/341578 ; urn:isbn:9789462955493 ; URN:NBN:NL:UI:10-1874-341578 ; https://dspace.library.uu.nl/handle/1874/341578

Chicago Manual of Style (16^th Edition):

Cabukusta, Birol. “Molecular Dissection of a Candidate Ceramide Sensor.” 2016. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/341578 ; URN:NBN:NL:UI:10-1874-341578 ; 1874/341578 ; urn:isbn:9789462955493 ; URN:NBN:NL:UI:10-1874-341578 ; https://dspace.library.uu.nl/handle/1874/341578.

MLA Handbook (7^th Edition):

Cabukusta, Birol. “Molecular Dissection of a Candidate Ceramide Sensor.” 2016. Web. 22 Jan 2021.

Vancouver:

Cabukusta B. Molecular Dissection of a Candidate Ceramide Sensor. [Internet] [Doctoral dissertation]. University Utrecht; 2016. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/341578 ; URN:NBN:NL:UI:10-1874-341578 ; 1874/341578 ; urn:isbn:9789462955493 ; URN:NBN:NL:UI:10-1874-341578 ; https://dspace.library.uu.nl/handle/1874/341578.

Council of Science Editors:

Cabukusta B. Molecular Dissection of a Candidate Ceramide Sensor. [Doctoral Dissertation]. University Utrecht; 2016. Available from: https://dspace.library.uu.nl/handle/1874/341578 ; URN:NBN:NL:UI:10-1874-341578 ; 1874/341578 ; urn:isbn:9789462955493 ; URN:NBN:NL:UI:10-1874-341578 ; https://dspace.library.uu.nl/handle/1874/341578

28. Wijten, P. A proteomics perspective at human circulating cells.

Degree: 2017, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/350727 ; URN:NBN:NL:UI:10-1874-350727 ; 1874/350727 ; urn:isbn:9789462956544 ; URN:NBN:NL:UI:10-1874-350727 ; https://dspace.library.uu.nl/handle/1874/350727

► This thesis describes both the development and the implementation of novel strategies of mass spectrometry based proteomics used to characterize and investigate human primary circulating… (more)

▼ This thesis describes both the development and the implementation of novel strategies of mass spectrometry based proteomics used to characterize and investigate human primary circulating immune cells. The studies were performed to gain better insights into protein constituents of these cells and to explore the option of using these cells for obtaining clinically relevant biomarkers. One of these projects involved finding potential biomarkers in circulating cells for coronary artery disease. Contrary to most protein based biomarker studies that target easily accessible patient material which are substantially hampered by dynamic range issues in protein abundance, we investigated circulating cells that should in theory hold disease state specific biochemical information with a more favorable dynamic range. This yielded a total of 2440 quantified proteins of which 62 proteins initially were revealed as significantly different. However upon closer inspection unavoidable and variable contamination of plasma, erythrocyte and leukocyte components became apparent, which could be traced back to the individual patient level. We presented that correlating the quantitative patient/control study with reported quantitative proteomes of potential contaminating cells allows for filtering of false positive biomarkers into the verification phase. Also, a novel method for identifying the platelet releasate was introduced based on a reversed releasate proteomics approach to unambiguously and quantitatively determine the platelets released from activated platelets against a resting platelet background. Using this strategy we were able to quantitatively discriminate the released proteins from uncontrolled lysis products. Monitoring the copy numbers of roughly 4500 platelet proteins it became apparent that following a full stimulation only 124 proteins were significantly released. The released proteins span a concentration range of at least 5 orders of magnitude. Secretion of several of these proteins were confirmed by ELISA analysis. The released proteins were highly enriched in exhibiting the known secretion tags, among them some known high abundant factors. Besides the well-known secreted proteins many novel low abundant proteins could be identified. The proteomes of neutrophil, eosinophil and basophil granulocytes are quantitatively characterized and compared. At present no comprehensive proteomes are available for these 3 granulocytes that are to some extent similar but have diverging roles as well. For each of these cell types a comprehensive proteome was identified and quantified, exposing many novel specific proteins for these three granulocyte types. A first step was taken to find the similarities between neutrophils, eosinophils and basophils, as well as to expose the proteins that may be involved in their diverging roles. Advisors/Committee Members: Heck, Albert, Scholten, Arjen.

Subjects/Keywords: Proteomics; Mass spectrometry; Biomarker; Platelet; Granulocyte; Neutrophil; Eosinophil; Basophil

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Wijten, P. (2017). A proteomics perspective at human circulating cells. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/350727 ; URN:NBN:NL:UI:10-1874-350727 ; 1874/350727 ; urn:isbn:9789462956544 ; URN:NBN:NL:UI:10-1874-350727 ; https://dspace.library.uu.nl/handle/1874/350727

Chicago Manual of Style (16^th Edition):

Wijten, P. “A proteomics perspective at human circulating cells.” 2017. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/350727 ; URN:NBN:NL:UI:10-1874-350727 ; 1874/350727 ; urn:isbn:9789462956544 ; URN:NBN:NL:UI:10-1874-350727 ; https://dspace.library.uu.nl/handle/1874/350727.

MLA Handbook (7^th Edition):

Wijten, P. “A proteomics perspective at human circulating cells.” 2017. Web. 22 Jan 2021.

Vancouver:

Wijten P. A proteomics perspective at human circulating cells. [Internet] [Doctoral dissertation]. University Utrecht; 2017. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/350727 ; URN:NBN:NL:UI:10-1874-350727 ; 1874/350727 ; urn:isbn:9789462956544 ; URN:NBN:NL:UI:10-1874-350727 ; https://dspace.library.uu.nl/handle/1874/350727.

Council of Science Editors:

Wijten P. A proteomics perspective at human circulating cells. [Doctoral Dissertation]. University Utrecht; 2017. Available from: https://dspace.library.uu.nl/handle/1874/350727 ; URN:NBN:NL:UI:10-1874-350727 ; 1874/350727 ; urn:isbn:9789462956544 ; URN:NBN:NL:UI:10-1874-350727 ; https://dspace.library.uu.nl/handle/1874/350727

29. Cristobal Gonzalez de Durana, A. Personalized proteomic profiles enabled by advances in mass spectrometry-based proteomics.

Degree: 2017, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/351052 ; URN:NBN:NL:UI:10-1874-351052 ; 1874/351052 ; urn:isbn:9789039367599 ; URN:NBN:NL:UI:10-1874-351052 ; https://dspace.library.uu.nl/handle/1874/351052

► The major aim of the work presented in this thesis was to generate personalized proteomics profiles by improving the chromatographic aspects of the proteomic experiment.… (more)

▼ The major aim of the work presented in this thesis was to generate personalized proteomics profiles by improving the chromatographic aspects of the proteomic experiment. In the first chapter an overview of proteomics is given and several practical aspects of a proteomic workflow are highlighted. An important aspect is the high complexity and the intrinsic dynamic range of the samples analyzed in proteomics. In order to reduce this complexity and perform a comprehensive study a separation strategy (involving one or more separation steps) is required. A detailed description of reversed phase chromatography is given in this chapter as is the most widely used chromatographic methodology in proteomics. Furthermore, an overview of mass spectrometers is given which includes details of both ionization sources and mass analyzers. Protein quantification is also described as in chapter 3 a quantitative proteomics workflow was applied. A separate section for intestinal stem cell biology describes the characteristics of adult stem cells, in general, and of intestinal stem cells, in particular. The impact of the identification of intestinal stem cells is also addressed and how this led to the development of organoids. Moreover, applications of organoids to investigate disease, colorectal cancer in this case, is further explored. In the second chapter we aimed to build an in-house ultra-high pressure liquid chromatography system with a high separation efficiency and resolution. An easy to implement design was constructed. Particles with small diameter (1.8 μm) were used in the stationary phase with the aim of obtaining an increased efficiency compared to conventionally used 3 μm particles. Thanks to advancements in HPLC system design, particularly with respect to generating and maintaining pressures, we were able to implement columns containing smaller particles (1.8 μm) in dimensions of 50 μm x 40 cm. Several flow rates were studied and gradients ranging from 23 to 458 minutes were optimized. The level of performance of this system was tested by coupling it to an Orbitrap Velos achieving over 4500 proteins with the longest analysis time. Furthermore, a mass spectrometer with faster sequencing speed was used (TripleTOF) which enables us to identify over 1400 proteins in a 23-minute gradient. In the third chapter we performed personalized proteome profiles of healthy and tumor human colon organoids. Peptides were extracted from the organoid samples and dimethyl labeled in order to obtain quantitative information. The optimized reversed phase chromatography described in chapter 2 was used here after SCX prefractionation, to perform a comprehensive proteomics study. Furthermore, two state-of-the art mass spectrometers were used which allowed us to perform optimal peptide sequencing for each SCX fraction. Proteins extracted from organoids grown from seven patients were analyzed and the data was combined with transcriptomics data. Although generic features of colorectal cancer could be obtained across all the patients, our data clearly… Advisors/Committee Members: Heck, Albert, Mohammed, Shabaz.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Cristobal Gonzalez de Durana, A. (2017). Personalized proteomic profiles enabled by advances in mass spectrometry-based proteomics. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/351052 ; URN:NBN:NL:UI:10-1874-351052 ; 1874/351052 ; urn:isbn:9789039367599 ; URN:NBN:NL:UI:10-1874-351052 ; https://dspace.library.uu.nl/handle/1874/351052

Chicago Manual of Style (16^th Edition):

Cristobal Gonzalez de Durana, A. “Personalized proteomic profiles enabled by advances in mass spectrometry-based proteomics.” 2017. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/351052 ; URN:NBN:NL:UI:10-1874-351052 ; 1874/351052 ; urn:isbn:9789039367599 ; URN:NBN:NL:UI:10-1874-351052 ; https://dspace.library.uu.nl/handle/1874/351052.

MLA Handbook (7^th Edition):

Cristobal Gonzalez de Durana, A. “Personalized proteomic profiles enabled by advances in mass spectrometry-based proteomics.” 2017. Web. 22 Jan 2021.

Vancouver:

Cristobal Gonzalez de Durana A. Personalized proteomic profiles enabled by advances in mass spectrometry-based proteomics. [Internet] [Doctoral dissertation]. University Utrecht; 2017. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/351052 ; URN:NBN:NL:UI:10-1874-351052 ; 1874/351052 ; urn:isbn:9789039367599 ; URN:NBN:NL:UI:10-1874-351052 ; https://dspace.library.uu.nl/handle/1874/351052.

Council of Science Editors:

Cristobal Gonzalez de Durana A. Personalized proteomic profiles enabled by advances in mass spectrometry-based proteomics. [Doctoral Dissertation]. University Utrecht; 2017. Available from: https://dspace.library.uu.nl/handle/1874/351052 ; URN:NBN:NL:UI:10-1874-351052 ; 1874/351052 ; urn:isbn:9789039367599 ; URN:NBN:NL:UI:10-1874-351052 ; https://dspace.library.uu.nl/handle/1874/351052

30. van de Waterbeemd, M.J. Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry.

Degree: 2017, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/356519 ; URN:NBN:NL:UI:10-1874-356519 ; 1874/356519 ; urn:isbn:9789462957343 ; URN:NBN:NL:UI:10-1874-356519 ; https://dspace.library.uu.nl/handle/1874/356519

► Native mass spectrometry and mass spectrometry in general, are powerful analytical tools for studying proteins and protein complexes. Native mass spectrometry may provide accurate mass… (more)

Subjects/Keywords: Mass spectrometry; Native mass spectrometry; Protein Complexes; Structural Biology; Viruses; Protein Nanocontainers

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APA (6^th Edition):

van de Waterbeemd, M. J. (2017). Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/356519 ; URN:NBN:NL:UI:10-1874-356519 ; 1874/356519 ; urn:isbn:9789462957343 ; URN:NBN:NL:UI:10-1874-356519 ; https://dspace.library.uu.nl/handle/1874/356519

Chicago Manual of Style (16^th Edition):

van de Waterbeemd, M J. “Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry.” 2017. Doctoral Dissertation, University Utrecht. Accessed January 22, 2021. https://dspace.library.uu.nl/handle/1874/356519 ; URN:NBN:NL:UI:10-1874-356519 ; 1874/356519 ; urn:isbn:9789462957343 ; URN:NBN:NL:UI:10-1874-356519 ; https://dspace.library.uu.nl/handle/1874/356519.

MLA Handbook (7^th Edition):

van de Waterbeemd, M J. “Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry.” 2017. Web. 22 Jan 2021.

Vancouver:

van de Waterbeemd MJ. Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry. [Internet] [Doctoral dissertation]. University Utrecht; 2017. [cited 2021 Jan 22]. Available from: https://dspace.library.uu.nl/handle/1874/356519 ; URN:NBN:NL:UI:10-1874-356519 ; 1874/356519 ; urn:isbn:9789462957343 ; URN:NBN:NL:UI:10-1874-356519 ; https://dspace.library.uu.nl/handle/1874/356519.

Council of Science Editors:

van de Waterbeemd MJ. Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry. [Doctoral Dissertation]. University Utrecht; 2017. Available from: https://dspace.library.uu.nl/handle/1874/356519 ; URN:NBN:NL:UI:10-1874-356519 ; 1874/356519 ; urn:isbn:9789462957343 ; URN:NBN:NL:UI:10-1874-356519 ; https://dspace.library.uu.nl/handle/1874/356519

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