Evaluating preparative isoelectric focusing of complex peptide mixtures for tandem mass spectrometry-based proteomics: a case study in profiling chromatin-enriched subcellular fractions in Saccharomyces cerevisiae.
We have evaluated the use of free-flow electrophoresis, an emerging separation method for preparative isoelectric focusing of complex peptide mixtures, as a tool for high-throughput tandem mass spectrometry-based proteomic analysis. In this study, we investigated the ability of free-flow electrophoresis to resolve and fractionate complex peptide mixtures and also the effectiveness of using peptide isoelectric point in conjunction with peptide match probability scoring in sequence database searching. As a model system for this study, we analyzed a chromatin-enriched fraction from the yeast Saccharomyces cerevisiae. This mixture was fractionated using preparative isoelectric focusing by free-flow electrophoresis, followed by online capillary liquid chromatography electrospray tandem mass spectrometry and sequence database searching. Our results demonstrate that (1) FFE effectively resolves and fractionates complex peptide mixtures on the basis of peptide isoelectric point and (2) the introduction of peptide pI is effective in minimizing both false positive and false negative sequence matches in sequence database searching of tandem mass spectrometry data.
Xie
Hongwei
H
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 321 Church Street SE, 6-155 Jackson Hall, Minneapolis, Minnesota 55455, USA.
Bandhakavi
Sricharan
S
Griffin
Timothy J
TJ
D000595:Amino Acid Sequence; D002843:Chromatin / Q000378:metabolism*; D030562:Databases, Protein; D004586:Electrophoresis / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002843:Chromatin; D010455:Peptides; D011506:Proteins