• ESI;
  • Ionization efficiency;
  • MALDI;
  • Peptide quantification;
  • Proteolytic cleavage;
  • Technology

A novel type of peptide standard is introduced that consists of two peptides combined in one synthetic molecule and separated by a proteolytic cleavage site. Upon enzymatic digestion, the two peptides are released in a molar one-to-one ratio. This method enables the generation of exact equimolar mixtures of two peptides of any nature and origin, thereby providing a valuable tool for the investigation of fundamental phenomena in MS. The applicability of the method is exemplified by the analysis of the effect of peptide sequence variations on the relative ionization efficiency in ESI- and MALDI-MS.