Expansion of the toolbox to decipher the (phospho)proteome

Abstract

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. We further found that many phosphopeptides identified with tandem SCX contain motives of basophilic kinases, emphasizing the importance of the identified phosphopeptide pools.