Labelling method to distinguish isobaric amino acids and amino acid combinations

A method for distinguishing isobaric amino acids and amino acid combinations of isoleucine and leucine;

• asparagine and glycine-glycine;
• glutamine and glycine-alanine;
• and/or glutamine and alanine-glycine, during polypeptide sequencing, the method comprising:
• digesting a polypeptide of interest with a plurality of proteases to produce shorter peptides, wherein the plurality of proteases is two or more of pepsin, asp-N, chymotrypsin, thermolysin, and Lys-N;
• reacting the produced peptides with a coupling reagent capable of derivatizing the free C-terminal carboxylic acid function of the peptides, under conditions to add a basic functional group having a positive charge;
• selecting a charge state of 2+ or more, and fragmenting the derivatized peptides in a mass spectrometer under conditions effective to generate at least w ions;
• and
• detecting said w ions by mass spectrometry, and identifying derivatized peptides which incorporate the additional mass of said basic functional group;
• wherein positive charges are added to the free C-terminal carboxylic acid function of the peptide and/or polypeptide, and said w ions are analyzed to distinguish between the isobaric amino acids and amino acid combinations of:
• isoleucine and leucine;
• asparagine and glycine-glycine;
• glutamine and glycine-alanine;
• and/or glutamine and alanine-glycine;
• and
• wherein said method achieves complete polypeptide sequencing while resolving all amino acid ambiguities.