Product(s) used in this publication: Reference Peptides for Targeted Proteomics - SpikeTides™ & SpikeMix™
Two important aspects of peptide and protein quantification by LC-MS/MS, the enzymatic digestion step and the internal standardization approach, were systematically investigated with a small protein, salmon calcitonin, which could be analyzed both without and with digestion. Quantification of undigested salmon calcitonin, after solid-phase extraction from plasma, resulted in a lower limit of quantification of 10 pg/mL, while introduction of a tryptic digestion step, followed by quantification of a signature peptide, increased this to 50 pg/mL. The sensitivity was reduced by interferences in the selected reaction monitoring (SRM) transition of the signature peptide due to the increase in sample complexity caused by the digestion and a less selective SRM transition of the signature peptide as compared to undigested salmon calcitonin. Eight internal standardization approaches were compared with respect to accuracy and precision in workflows with and without digestion. Analogue and stable-isotope-labeled (SIL) internal standards were evaluated including an in-house created (18)O-labeled peptide, a cleavable SIL peptide, and an internal standard created by differential derivatization of the signature peptide. We conclude that the best internal standard for the workflows both with and without digestion was the SIL form of the analyte, although the use of several SIL signature peptides and a differentially derivatized signature peptide also resulted in methods with performances which meet the FDA guidelines.