Recommendations for Peptides Used In Mass Spectrometry Assays

Clinical laboratories are beginning to employ bottom-up MS-based methods with stable isotope-labeled standards for protein quantitation of biosamples, yet the procedures for development (from peptide selection to handling) and analysis are not well standardized. This report provides a compendium of guidelines and specifications from experts in the field for effectively using internal peptide standards to quantify endogenous peptides in complex proteolytic sample digests. The goal of the recommendations is to achieve precise, relative quantitation that can be harmonized across laboratories with high a degree of repeatability/reproducibility.

Abstract

For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays.

The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

Hoofnagle AN, Whiteaker JR, Carr SA, Kuhn E, et al.

Read More