Yang Xiaofeng, Wu Huaping, Kobayashi Tomoyoshi, Solaro R John, van Breemen Richard B
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Anal Chem. 2004 Mar 1;76(5):1532-6. doi: 10.1021/ac035203v.
Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.
尽管α-氰基-4-羟基肉桂酸作为一种出色的基质，可用于使用基质辅助激光解吸/电离飞行时间（MALDI TOF）质谱分析大多数肽段，但磷酸化肽段的电离通常会受到非磷酸化肽段的抑制。作为一种替代基质，发现2',4',6'-三羟基苯乙酮（THAP）与柠檬酸二铵可克服此问题，用于磷酸化蛋白质酶解产物的MALDI TOF质谱分析。具体而言，在正离子MALDI TOF质谱分析中，使用THAP时，牛β-酪蛋白和蛋白激酶C（PKC）处理的小鼠心肌肌钙蛋白I的胰蛋白酶消化物中磷酸化肽段的丰度提高了10倍以上。磷酸化肽段的质子化分子足够丰富，以至于使用源后衰变TOF质谱来确认每个肽段中的磷酸基团数量。最后，胰蛋白酶消化后，以THAP作为基质进行MALDI TOF质谱分析，有助于鉴定PKC处理的肌钙蛋白I中的一个独特磷酸化位点。
