Advancing liquid chromatography- mass spectrometry based technologies for proteome research

Abstract

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 important in the maintenance of the pluripotency state of hESCs. Several hundreds of Tyr phosphorylation sites could be identified and quantified of which some 140 showed a differential regulation upon FGF-2 stimulation. These regulated sites were found on proteins that include FGF receptors, members of the Src family, proteins involved in actin polymerization and cyclin dependent kinases.