Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation directing moiety

We describe a method for comparative quantitation and de novo peptide seque ncing of proteins separated either by standard chromatographic methods or b y one- and two-dimensional polyacrylamide gel electrophoresis. The approach is based on the use of an isotopically labeled reagent to quantitate (by m ass spectrometry) the ratio of peptides from digests of a protein being exp ressed under different conditions. The method allows quantitation of the ch anges occurring in spots or bands that contain more than one protein and ha s a greater dynamic range than most staining methods. Since the reagent car ries a fixed positive charge under acidic conditions and labels only the N- terminal of peptides, the interpretation of tandem mass spectra to obtain s equence information is greatly simplified. The sequences can easily be extr acted for homology searches instead of using indirect mass spectral-based s earches and are independent of posttranslational modifications.