Direct Ampholyte-Free Liquid-Phase Isoelectric Peptide Focusing: Application to the Human Serum Proteome
In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (microLC)-MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte-free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.
Blood proteins, humans, isoelectric focusing, mass spectrometry, peptide fragments, proteome, proteomics
Xiao, Zhen; Conrads, Thomas P.; Lucas, David A.; Janini, George M.; Schaefer, Carl F.; Buetow, Kenneth H.; Issaq, Haleem J.; and Veenstra, Timothy D., "Direct Ampholyte-Free Liquid-Phase Isoelectric Peptide Focusing: Application to the Human Serum Proteome" (2004). Pharmaceutical Sciences Faculty Publications. 476.