Split-field drift tube/mass spectrometry and isotopic labeling techniques for determination of single amino acid...

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.