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We report here a method for the identification of free or blocked N-terminal peptide of in-gel digested isolated proteins. The primary amino groups of the gel-entrapped protein are blocked with normal acetic or succinic anhydride, and the protein is digested with a high-specificity protease. The generated peptides are treated with an equimolar mixture of normal and deuterated acetic anhydride. Upon mass spectrometric analysis internal peptides display a complex isotopic ion distribution while the N-terminal peptide shows a normal isotopic ion distribution. The procedure was applied to the identification of the N-terminus of individual and protein mixtures isolated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE)., (Copyright 2007 John Wiley & Sons, Ltd.)

Mass spectrometry is now firmly established as a powerful technique for the identification and characterization of proteins when used in conjunction with sequence databases. Various approaches involving stable-isotope labeling have been developed for quantitative comparisons between paired samples in proteomic expression analysis by mass spectrometry. However, interpretation of such mass spectra is far from being fully automated, mainly due to the difficulty of analyzing complex patterns resulting from the overlap of multiple peaks arising from the assortment of natural isotopes. In order to facilitate the interpretation of a complex mass spectrum of such a mixture, such as an MS spectrum of a stable-isotope-enriched ion species, we report on the development of a software application, 'Matching' (web accessible), that enables the automatic matching of theoretical isotope envelopes to multiple ion peaks in a raw spectrum. It is particularly useful for resolving the relative abundances of narrow-split paired peaks caused by enrichment with a stable isotope, such as 18O, 13C, 2H, or 15N., (2004 John Wiley & Sons, Ltd.)

SeqMS, a software program designed for the automated interpretation of high-energy collision-induced dissociation (CID) mass spectra of singly protonated peptides ionized by fast atom bombardment, has been developed. The software is capable of probing the sequence of an unknown peptide, and even of certain modified peptides. The program, compiled for WINDOWS95 or NT, also permits the retrieval of raw data and the reconstruction of the spectra on a user-friendly graphical interface with the aid of several tools for processing the spectra, which include setting multiple threshold levels and automatic peak detection. SeqMS is capable of generating candidate sequences, based on the detected peaks, and of displaying the resulting assignments for each candidate in a spectrum or in tabular form. The software has the following capabilities: 1) the ions derived from backbone and side-chain fragmentations, internal and immonium ions, and side-chain loss ions can be used for calculation; 2) 18O-labeling of a peptide at the C terminus, a methodology which was developed to differentiate N-terminal from C-terminal ions, is applicable as an optional setting; 3) modified amino acids and N- or C-terminal blocking groups are taken into account for calculation according to the user's setting in a library; 4) amino acid composition and partial or complete amino acid sequence of a peptide can be used as input for calculation; 5) the assignments of signal output in a spectrum can be graphically edited, and then re-calculated based on the edited peaks. The efficacy of the program is demonstrated by testing 74 high-energy CID spectra, obtained using a four-sector instrument, of synthetic, proteolytic, and biologically active peptides, some of which contain modified groups.