Proteomics Department, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba.
Anal Chem. 2010 Oct 15;82(20):8492-501. doi: 10.1021/ac1012738.
Edman degradation in the gas phase has been observed by collision activated dissociation of N-terminal phenylthiocarbamoyl (PTC) protonated peptide to yield abundant complementary b₁ and y(n-1) ion pairs. Here, we demonstrated the relation between the observed losses of aniline and/or the entire PTC derivatizing group with the availability of mobile protons using electrospray ionization mass spectrometry. In order to select the peptides with more efficient fragmentation, while simplifying the mixture of peptides, we extend the phenylisotiocyanate (PITC) derivatization of amino groups to the selective isolation of multiply charged peptides (those having the number of arginines and histidines residues higher than one) using a procedure previously developed in our group. Thus, it was possible to identify in the filtered protein database the sequence of the isolated multiply charged peptides derived from a single protein and a complex mixture of proteins extracted from Escherichia coli using only the molecular mass and the N-terminal amino acid information. For this purpose, we developed a novel bioinformatic tool for automatic identification of peptides from liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments, which potentially can be used in high-throughput proteomics.
气相 Edman 降解已通过碰撞活化裂解 N 端苯硫代羰酰基（PTC）质子化肽得到了广泛的互补 b₁ 和 y(n-1)离子对而被观察到。在这里，我们使用电喷雾电离质谱证明了观察到的苯胺和/或整个 PTC 衍生基团的损失与可移动质子之间的关系。为了选择具有更高效率碎片的肽，同时简化肽混合物，我们扩展了苯异硫氰酸酯（PITC）衍生化氨基的方法，以选择性地分离多电荷肽（具有一个以上精氨酸和组氨酸残基的肽），该方法是我们小组之前开发的。因此，仅使用分子量和 N 端氨基酸信息，就可以从单个蛋白质和从大肠杆菌提取的复杂蛋白质混合物的过滤蛋白质数据库中鉴定出从分离的多电荷肽衍生的序列。为此，我们开发了一种用于自动鉴定液相色谱-串联质谱（LC-MS/MS）实验中肽的新型生物信息学工具，该工具可用于高通量蛋白质组学。
