Labelled compounds and methods for mass spectrometry-based quantification

A method for selecting the label and label position of at least one suitable reference peptide for use in a method for the absolute or relative quantitative analysis of at least one of proteins or peptides, in each case with or without post translational modification(s), using a mass spectrometry method comprising:

• a first step where unlabeled proteins from an endogenous mixture are digested and subsequently digestion products thereof selected,
• a second step where said digestion products are fragmented, and
• a third step where a combined fragment spectrum is acquired comprising b-ions as well as y-ions of said digestion products,
• wherein at least one reference peptide with or without post translational modification(s) is added to said mixture before or after digestion or both, is fragmented, acquired, and stored in said combined fragment spectrum comprising also b-ions and y-ions of said digestion products,
• wherein said at least one reference peptide is added in a known concentration in case of absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis,
• wherein said at least one reference peptide 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
• wherein the position of the label at the C-terminus, or within the four terminal amino acids at the C-terminus, or the position of the label at the N-terminus, or within the four terminal amino acids at the N-terminus, or both, is selected using a procedure which takes into account at least one of the availability of the labeled version of the corresponding amino acid at the respective position or the complexity of the incorporation of the labeled version of the corresponding amino acid at the respective position, and wherein the label and label position is selected so as to be optimized with respect to these parameters.