title: Improved detection of multi-phosphorylated peptides by LC-MS/MS without phosphopeptide enrichment.
abstract: Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi- phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.
authors.affiliation: Department of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
authors.forename: Suwha
authors.initials: S
authors.lastname: Kim
authors.forename: Hyunwoo
authors.initials: H
authors.lastname: Choi
authors.forename: Zee-Yong
authors.initials: ZY
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D004526:Efficiency; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D010455:Peptides; D010748:Phosphopeptides; D014357:Trypsin; D019286:Endopeptidase K