Keough T, Lacey M P, Fieno A M, Grant R A, Sun Y, Bauer M D, Begley K B
The Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, OH, USA.
Electrophoresis. 2000 Jun;21(11):2252-65. doi: 10.1002/1522-2683(20000601)21:11<2252::AID-ELPS2252>3.0.CO;2-O.
Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.
已开发出优化程序，用于将磺酸基团添加到低水平肽的N端。这些程序已应用于通过二维（2-D）凝胶电泳分离的蛋白质经胰蛋白酶消化产生的肽。使用基质辅助激光解吸/电离（MALDI）源后衰变（PSD）和电喷雾电离串联质谱法对衍生化肽进行测序。从亚皮摩尔量的蛋白质开始，已获得可靠的PSD测序结果。我们估计目前凝胶中蛋白质的PSD测序极限约为300 fmol。衍生化肽的PSD质谱通常比未衍生化时获得的质谱允许更特异的蛋白质序列数据库搜索。我们还报告了这些新型肽衍生物的初始自动电喷雾电离串联质谱测序。两种类型的串联质谱都为精氨酸末端肽提供了可预测的裂解模式。这些谱很容易从头解释，并且有助于对其序列已输入数据库的蛋白质进行容错鉴定。
