A software program to aid peptide sequencing based on MS/MS data is of quite importance since MS/MS has become one of the key tools for sequencing of many biologically important proteins. Here we present "SeqMS", a software program designed for automated interpretation of high-energy collision-induced dissociation (CID) spectra of peptides, which has been developed based on a previous MS-DOS program [1]. The program, compiled for WINDOWS'95, allows easy retrieval of raw data files, peak detection with multiple threshold levels and automatic assignments of signals to amino acid sequence with a comfortable and powerful graphical user interface. The software has the following computational capabilities: 1) the ions derived from backbone and side-chain fragmentation, internal and immonium ions, and side-chain lost ions can be used for calculation; 2) 18O-labeling of a peptide at the C terminus, the methodology which was developed to differentiate the N-terminal ions from the C-terminal ones [2], is available as an optional setting; 3) modified amino acids, or the presence of N- or C-terminal blocking groups are taken into account for calculation according to the user's setting in a library; 4) a partial sequence or amino acid composition of a peptide, if available, can be input and used for calculation. SeqMS has been used for the interpretation of product ion spectra of peptides, obtained by high-energy CID using two- or four-sector instruments. The efficacy of the software for sequencing was demonstrated by applying it to natural and synthetic peptides, some of which contain modifications on amino acids.
To illustrate the utility of 18O-labeling of a peptide to sequencing with SeqMS, a peptide giving overlapped product ion signals in MS/MS, but which could be differentiated after 18O-labeling at the C-terminal carboxyl group, was examined. The two MS/MS spectra (top), retrieved by SeqMS, were obtained from MH+ and (M+3)+ observed for the partially 18O-labeled peptide (Gly-Asp-Ser-His-Asp-Pro-Gly) [3]. The y and b ions generated by the loss of two amino acid residues from the N and C terminus, respectively, completely overlapped at m/z=512.2. Thus, the y and b ions below 512.2 could not be definitely differentiated from each other, giving two possible sequences: one has -Ser-His-Asp-, and the other, -Asp-His-Ser-. The result obtained by using only the product ion spectrum from MH+ gave both real and retro-sequence at the 4th and 7th position, respectively, among the 15 candidates (left in the middle), and several heterogeneous incorrect sequences. On the other hand, the real sequence was obtained with the relatively higher score at the first position by applying both MS/MS spectra from MH+ and (M+3)+ (right in the middle and bottom spectrum, which are the outputs from SeqMS).
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