Li Wei, Boykins Robert A, Backlund Peter S, Wang Guiyu, Chen Hao-Chia
Endocrinology and Reproduction Research Branch, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Anal Chem. 2002 Nov 15;74(22):5701-10. doi: 10.1021/ac020259v.
Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.
串联质谱长期以来一直是确定蛋白质中磷酸化位点的固有工具。然而，在低能碰撞诱导解离过程中，磷酸丝氨酸和磷酸苏氨酸残基的磷酸部分丢失是一种常见现象，这使得磷酸丝氨酸（P-Ser）和磷酸苏氨酸（P-Thr）残基的鉴定变得复杂。研究了一种通过β-消除/亚硫酸盐添加将-HPO4转化为-SO3后，利用电喷雾串联质谱直接对丝氨酸和苏氨酸磷酸化位点进行测序的方法。五种模型磷酸肽，包括三种合成的含P-Ser、P-Thr或同时含P-Ser和P-Thr的肽；一种蛋白激酶C磷酸化的肽；以及一种源自β-酪蛋白胰蛋白酶消化产物的磷酸肽，经过修饰后，使用电喷雾四极杆离子阱质谱仪进行测序。通过氨基酸分析发现，将含P-Ser或P-Thr的肽与Na2SO3/NaOH孵育后，分别有90%的P-Ser和80%的P-Thr转化为半胱氨酸磺酸和β-甲基半胱氨酸磺酸。这种转化可以在1 microM的肽浓度下进行。在适用于普通肽的串联质谱测序的一般条件下，序列中的半胱氨酸磺酸和β-甲基半胱氨酸磺酸残基都显示出稳定性且易于识别。
