Valentine Stephen J, Sevugarajan S, Kurulugama Ruwan T, Koeniger Stormy L, Merenbloom Samuel I, Bohrer Brian C, Clemmer David E
Predictive Physiology and Medicine, 1424 West Adams Hill Circle, Bloomington, Indiana 47403, USA.
J Proteome Res. 2006 Aug;5(8):1879-87. doi: 10.1021/pr060068z.
A combination of split-field drift tube/mass spectrometry and isotopic labeling techniques is evaluated as a means of identifying single amino acid polymorphisms (SAAPs) in proteins. The method is demonstrated using cytochromec (equine and bovine) and hemoglobin (bovine and sheep). For these studies, proteins from different species are digested with trypsin, and the peptides are labeled at primary amine groups [using either a light (H(3))- or heavy (D(3))-isotopic reagent]. SAAP analysis is carried out by mixing the light-labeled peptides of one species with the heavy-labeled peptides of the other and electrospraying the resulting mixture into a split-field drift tube/mass spectrometer. Peptides having the same sequence in both species appear as doublets in the mass spectrum [shifted in mass-to-charge (m/z) according to the number of incorporated labels]; additionally, these species have identical mobility distributions. Peptides having sequences that differ by one amino acid appear as peaks in the mass spectrum that are shifted in m/z according to the mass difference associated with the SAAP and the number of incorporated labels. The ion mobility distributions for these peptides (differing by only a single amino acid) can often be rationalized by their expected similarities or differences providing additional evidence that they are related. In all, 12 and 26 peptide variants (between species) corresponding to 5 and 11 amino acid polymorphisms have been identified for the cytochrome c and hemoglobin protein samples, respectively.
评价了裂场漂移管/质谱联用和同位素标记技术相结合的方法，作为鉴定蛋白质中单个氨基酸多态性（SAAP）的手段。使用细胞色素c（马和牛）和血红蛋白（牛和羊）对该方法进行了验证。在这些研究中，用胰蛋白酶消化来自不同物种的蛋白质，并用轻（H(3)）或重（D(3)）同位素试剂标记肽段的伯胺基团。通过将一种物种的轻标记肽段与另一种物种的重标记肽段混合，并将所得混合物电喷雾到裂场漂移管/质谱仪中进行SAAP分析。在两个物种中具有相同序列的肽段在质谱图中表现为双峰[根据掺入标记的数量在质荷比（m/z）上发生偏移]；此外，这些物种具有相同的迁移率分布。序列相差一个氨基酸的肽段在质谱图中表现为峰，其m/z根据与SAAP相关的质量差异和掺入标记的数量而发生偏移。这些仅相差一个氨基酸的肽段的离子迁移率分布通常可以通过它们预期的相似性或差异来解释，这提供了它们相关的额外证据。对于细胞色素c和血红蛋白蛋白质样品，分别鉴定出了与5个和11个氨基酸多态性相对应的12个和26个肽段变体（物种间）。
