title: Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome.
abstract: 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.
authors.affiliation: Laboratory of Proteomics and Analytical Technologies, National Cancer Institute at Frederick, SAIC-Frederick, Frederick, MD 21702, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xiao
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.forename: David A
authors.initials: DA
authors.lastname: Lucas
authors.forename: George M
authors.initials: GM
authors.lastname: Janini
authors.forename: Carl F
authors.initials: CF
authors.lastname: Schaefer
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Buetow
authors.forename: Haleem J
authors.initials: HJ
authors.lastname: Issaq
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D020543:Proteome