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 sequencing of proteins separated either by standard chromatographic methods or by one- and two-dimensional polyacrylamide gel electrophoresis. The approach is based on the use of an isotopically labeled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. The method allows quantitation of the changes occurring in spots or bands that contain more than one protein and has a greater dynamic range than most staining methods. Since the reagent carries a fixed positive charge under acidic conditions and labels only the N-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. The sequences can easily be extracted for homology searches instead of using indirect mass spectral-based searches and are independent of posttranslational modifications.
Münchbach
M
M
Protein Chemistry, Swiss Federal Institute of Technology, Rüschlikon.
Quadroni
M
M
Miotto
G
G
James
P
P
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis*; D020539:Sequence Analysis, Protein
D011506:Proteins