Chemical labelling methods for gel free based proteomics

Abstract: In this thesis different aspects of gel free procedures for mass spectrometry based proteome analysis are addressed, with emphasis on labelling techniques. It is based on three original articles where the effects imposed onto peptides upon modification are investigated, and the development of a new type of labelling strategy is described. Chemical modification has been of great importance for the peptide based proteomics approach, as they provide a way to impose desired properties onto the peptides. One possibility is to improve ionisation efficiency by introducing an easily protonated entity. By strategic placement of such a label, favoured ionisation of certain fragments upon MS/MS analysis can work to simplify the fragmentation spectra, and consequently facilitate their interpretation. In this work, such a charge-directing fragmentation label and its effects using a new type of mass spectrometer was investigated. One of the main applications for the various labelling techniques however, has been the ability to produce isotopic counterparts of peptides, which enables quantification of the sample constituents. Several procedures to accomplish this, using a wide assortment of labels have, and are being developed. The Parent Ion Quantitation Scanning (PIQS) method described in this work, was developed aiming at more efficient global analysis of protein expression levels. The method was constructed to only sample peptides from differentially expressed proteins, thereby alleviating the large amounts of data usually generated, and only targeting relevant information, when aiming for analysis of differences in protein abundance.