Choi Hyunwoo, Lee Hye-suk, Park Zee-Yong
Department of Life Sciences, Gwangju Institute of Science & Technology 1 Oryong-Dong, Buk-Gu, Gwangju, Korea 500-712.
Anal Chem. 2008 Apr 15;80(8):3007-15. doi: 10.1021/ac7023393. Epub 2008 Mar 18.
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.).
本文展示了一种改进的方法，用于在低pH条件（pH约为1.7，含3%甲酸）下通过反相液相色谱-串联质谱（RPLC-MS/MS）分析检测多磷酸化肽段，以牛α-酪蛋白和β-酪蛋白这两种模型磷蛋白为例。pH条件从正常（pH约为3.0，含0.1%甲酸）变为低pH（pH约为1.7，含3%甲酸）时，显著提高了携带负（-）溶液电荷状态的多磷酸化肽段的检测限。特别是，在低pH条件下，仅加载50 fmol经胰蛋白酶消化的牛β-酪蛋白就能检测到牛β-酪蛋白四磷酸化肽段，这比在正常pH条件下检测该肽段所需的量低200倍。为了理解低pH的影响，在两种不同pH条件下，通过RPLC-MS/MS分析对不同加载量的经胰蛋白酶消化的牛α-酪蛋白和β-酪蛋白进行了分析。由于无法通过肽合成轻易获得合成多磷酸化肽段，因此在本研究中无法证明低pH条件是否通过提高电离效率来改善多磷酸化肽段的检测。有趣的是，多次磷酸化事件导致的亲水性增加对肽段在反相柱材料上的保留产生了负面影响。研究还表明，低pH条件可以有效增强多磷酸化肽段在反相柱材料上的保留。使用从小鼠脑组织制备的实际富含磷酸肽的样品测试了低pH RPLC分析的实用性。此前，低pH溶剂已用于强阳离子交换（SCX）分级分离和二氧化钛富集过程，以便在磷酸肽富集程序中选择性富集磷酸肽，但此前尚未报道在低pH条件下的RPLC-MS/MS分析中多磷酸化肽段检测的改善情况（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.）。
