Improved Detection of Multi-phosphorylated Peptides by LC-MS/MS without Phosphopeptide Enrichment

  • Kim, Suwha (Department of Life Sciences, Gwangju Institute of Science and Technology) ;
  • Choi, Hyunwoo (Department of Life Sciences, Gwangju Institute of Science and Technology) ;
  • Park, Zee-Yong (Department of Life Sciences, Gwangju Institute of Science and Technology)
  • Received : 2007.01.12
  • Accepted : 2007.03.06
  • Published : 2007.06.30

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.

Keywords

LC-MS;Multi-Phosphorylated Peptides

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