Towards The Understanding of Hydrostatic Pressure Effects on Proteomic Sample Preparation Methods

摘要

PCT-based extraction of complex proteomic samples results in selective enrichment of membrane-associated proteins. Pressure acts synergistically with chaotropic agents and organic solvents on many proteins, particularly useful in less stringent extraction buffers that are more compatible with downstream chromatography and mass spectrometry analysis. The data also suggest that pressure effects on digestion efficiency are substrate protein-specific. Pressure does not appear to negatively affect digestion of "easy" protein substrates (no significant decrease in peptide recovery or increase in number of semitryptic or miscleaved peptides). Moreover, pressure digestion sheds additional light on the "The Dark Side of the Proteome" by providing significant improvements in quantitative recovery of peptides for those proteins that are otherwise resistant to trypsin digestion. Further investigation of this hypothesis is currently being conducted using model integral membrane proteins and complex proteomic samples such as protein extracts from adipose tissue.