The invention relates to methods for making a peptide standard for mass spectrometry said method comprising (a) identifying endopeptidase cleavage sites in a parent polypeptide sequence of interest (b) selecting peptide sequences from said parent polypeptide which are defined by endopeptidase cleavage sites of step (a) (c) adding a C-terminal extension to each selected sequence wherein if the endopeptidase cleavage site is C-terminal to its recognition sequence then the C-terminal extension comprises 1 to 6 amino acids, wherein if the endopeptidase cleavage site is N-terminal to its recognition sequence then the C-terminal extension comprises said recognition sequence, wherein if the endopeptidase cleavage site is within its recognition sequence then the C-terminal extension comprises the remainder of said recognition sequence C-terminal to the cleavage site and (d) synthesizing a peptide having the extended amino acid sequence of step (c). The invention also relates to peptide standards and methods of analysing samples.
