title: Detection of multiphosphorylated peptides in LC-MS/MS analysis under low pH conditions.
abstract: An improved method of detection of multiphosphorylated peptides by RPLC-MS/MS analysis under low pH conditions (pH approximately 1.7, 3% formic acid) is demonstrated for the model phosphoproteins, bovine alpha- and beta-casein. Changes in the pH conditions from normal (pH approximately 3.0, 0.1% formic acid) to low (pH approximately 1.7, 3% formic acid) significantly improved the detection limit of multiphosphorylated peptides carrying negative (-) solution charge states. In particular, bovine beta-casein tetraphosphorylated peptide, was detected with a loading amount of only 50 fmol of trypsin-digested bovine beta-casein under low pH conditions, which is 200 times lower than necessary to detect the peptide under normal pH conditions. In order to understand the low pH effect, various loading amounts of trypsin-digested bovine alpha- and beta-caseins were analyzed by RPLC-MS/MS analyses under two different pH conditions. The question of whether the low pH condition improves the detection of multiphosphorylated peptides by increasing ionization efficiencies could not be proven in this study because synthetic multiphosphorylated peptides could not be easily obtained by peptide synthesis. Interestingly, increased hydrophilicity resulting from multiple phosphorylation events is shown to negatively affect the peptide retention on reversed-phase column material. It was also demonstrated that the low pH condition could effectively enhance the retention of multiphosphorylated peptides on reversed-phase column material. The usefulness of low pH RPLC analysis was tested using an actual phosphopeptide-enriched sample prepared from mouse brain tissues. Previously, low pH solvents have been used in SCX fractionation and TiO2 enrichment processes to selectively enrich phosphopeptides during the phosphopeptide enrichment procedure, but the improved detection of multiphosphorylated peptides in RPLC-MS/MS analysis under low pH conditions has not been reported before (Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093-1101. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1488-1493. Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243-5250.).
authors.affiliation: Department of Life Sciences, Gwangju Institute of Science & Technology 1 Oryong-Dong, Buk-Gu, Gwangju, Korea 500-712.
authors.forename: Hyunwoo
authors.initials: H
authors.lastname: Choi
authors.forename: Hye-suk
authors.initials: HS
authors.lastname: Lee
authors.forename: Zee-Yong
authors.initials: ZY
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002021:Buffers; D002364:Caseins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D005561:Formates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis*; D010756:Phosphoric Acids / Q000737:chemistry; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002021:Buffers; D002364:Caseins; D005561:Formates; D010750:Phosphoproteins; D010756:Phosphoric Acids; C030544:formic acid; C030242:phosphoric acid