Labelled compounds and methods for mass spectrometry-based quantification

A method for the absolute or relative quantitative analysis of proteins and/or peptides with or without post translational modification(s) using a mass spectrometry method in which:

• in a first step unlabeled proteins from an endogenous mixture are digested and subsequently digestion products thereof selected,
• in a second step said digestion products are fragmented, and
• in a third step a combined fragment spectrum is acquired comprising b-ions and y-ions of said digestion products,
• wherein at least one reference peptide is added to said mixture before and/or after digestion, is fragmented, acquired, and stored in said combined fragment spectrum comprising also b-ions and y-ions of said digestion products,
• wherein the said at least one reference peptide is added in a known concentration for absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis,
• wherein said at least one reference peptide this is selectively isotopically labeled by having incorporated:
• one isotopically labeled amino acid forming its very C-terminus or being one of the four terminal amino acids at the C-terminus and
• one isotopically labeled amino acid forming its very N-terminus, or being one of the four terminal amino acids at the N-terminus, and
• wherein the isotopically labeled amino acids are unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety, wherein said unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety comprise one or more atoms that are isotopically labeled such that said one or more atoms are present in the amino acid and not in the chemically modifying moiety.