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Chemical labeling for the analysis of proteins, peptides and metabolites bij mass spectrometry

11 May 2009

PhD ceremony: N.J-R. Abello, 13.15 uur, Academiegebouw, Broerstraat 5, Groningen

Thesis: Chemical labeling for the analysis of proteins, peptides and metabolites bij mass spectrometry

Promotor(s): prof. R.P.H. Bischoff, prof. D.S. Postma

Faculty: Mathematics and Natural Sciences

 

This thesis reports techniques for the analysis of proteins, peptides and metabolites by LC-MS with a strong emphasis on chemical labeling for quantification or affinity enrichment. Tyrosine nitration is a low abundance post-translational protein modification reviewed in detail in chapter 2, and Chapter 5 presents an improved technique for the enrichment and the analysis of nitrotyrosine-containing peptides, based on the complete blocking of all primary amines followed by the conversion of nitrotyrosine to aminotyrosine and the selective biotinylation of the latter. The entire reaction sequence is performed in a single buffer with no need for sample cleanup or pH changes. Chapter 4 reports the application of a new poly(ethylene glycol) (PEG)-based method for stable isotope labeling of primary-amine-containing compounds such as amino acids and glutathione, using 13C-contanining pentafluorophenyl-activated esters of PEG derivatives (PEG-OPFP). This labeling results in improved LC retention and MS detection. Chapters 4 and 5 use a method described in Chapter 3, i.e. a generic approach to increase the selectivity of primary amine derivatization with activated carboxylic acid esters such as N-hydroxysuccinimide (NHS) esters. This technique is based on the incubation of samples in a boiling water bath, with no requirement for subsequent pH correction or sample cleanup. Finally, Chapter 6 investigates the degradation of elastin, a structural insoluble protein that gives elasticity to tissues and organs, under the action of proteases from different families. It establishes that LC-MS is a powerful technique to study elastin-derived peptides, even though their study remains an analytical challenge.

 

 

Last modified:15 September 2017 3.38 p.m.
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