Characterization of Platelet Proteins Using Peptide Centric Proteomics

Summary

In modern proteomics, undersampling of low abundant, cumbersome, and hydrophobic proteins states one of the major problems. To overcome this, especially in two 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) eminent drawbacks, the so-called peptide-centric techniques have been developed. These approaches do not separate proteins prior to digestion, but instead proteolytically generate peptide mixtures after it. However, by this procedure already complex protein mixtures become even more extensive peptide mixtures. Particularly, when dealing with large proteomes, the generated sample complexity is vast and therefore difficult to analyze. When separated and analyzed by LC/MS, too many peptides may enter the mass spectrometer at a certain time point, and only a small fraction of ions is selected for subsequent MS/MS analysis. Although protein hydrophobicity and size play minor roles (as long as protease cleavage sites are accessible), low copy number can severely limit identification rates. To reduce the amount of peptides entering the mass spectrometer simultaneously without the loss of overall proteomic information, different techniques have been developed. Among these, an approach is represented by COFRADIC (Combined Fractional Diagonal Chromatography).

COFRADIC is a chromatography-based technique enabling the sorting of peptides due to retention time shifts after a specific modification step. In the original approach, a complex peptide mixture is separated by a primary RP-HPLC (reversed-phase high-performance liquid chromatography) run and fractions are retained. Subsequently, these fractions are modified to specifically change retention times of peptides and separated in one or more secondary RP-HPLC runs. In this chapter, COFRADIC approaches for methionine or cysteine containing as well as N-terminal peptides are described.

Besides the reduction of sample complexity, the major advantage of COFRADIC might be seen in its versatility. Nearly every feature unique for a subset of peptides, which can be specifically modified by a sorting reaction, is accessible for COFRADIC. Among these are protein phosphorylation, N-glycosylation, and in vivo protein processing sites. Finally, COFRADIC allows the analysis of large numbers of samples and is highly automatable.