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.
Xiao
Zhen
Z
Laboratory of Proteomics and Analytical Technologies, National Cancer Institute at Frederick, SAIC-Frederick, Frederick, MD 21702, USA.
Conrads
Thomas P
TP
Lucas
David A
DA
Janini
George M
GM
Schaefer
Carl F
CF
Buetow
Kenneth H
KH
Issaq
Haleem J
HJ
Veenstra
Timothy D
TD
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
D001798:Blood Proteins; D010446:Peptide Fragments; D020543:Proteome