(53b) a High-Throughput Information Added Proteomic Separation Strategy Using Free Flow Electrophoresis and Tandem Mass Spectrometry

One challenge of mass spectrometry based proteomics is to identify comprehensively proteins with high confidence from cells, tissues and other complex biological sources. To this end, we have developed the use of Free Flow Electrophoresis (FFE) for preparative isoelectric focusing (IEF) of complex peptide mixtures as a tool for high-throughput tandem mass spectrometry-based proteomic analysis. In this strategy, peptides are fractionated by FFE in the first separation dimension, followed by capillary liquid chromatography, tandem mass spectrometry and sequence database searching. We have termed the use of FFE separations of peptide mixtures as an ?information-added? separation approach, as it not only provides a high resolution separation, but also it adds a constraint of peptide isoelectric point to the determination of peptide sequence matches in the sequence database search of the MS/MS data. We have demonstrated the effectiveness of FFE to resolve and fractionate a complex peptide mixture from the digestion of enriched chromatin-associated proteins in S. cerevisae and the power of using peptide isoelectric point (pI) as an additional constraint in protein identification by sequence database searching. In more recent work, we have applied the strategy to profile proteins in human saliva and obtained a comprehensive catalogue of proteins from this complex bodily fluid, which further demonstrates the effectiveness of FFE peptide separation as general and powerful tool for mass spectrometry based proteomics.