Utilizing enzymatic digestion procedures in the bioanalytical laboratory

Abstract

In recent years, the use of LC–MS technologies in the bioanlytical laboratory for quantitation of peptide/protein biomarkers and biotherapeutics has increased dramatically. The increased interest is due to the improvement in sensitivity of MS instruments over the last 5–10 years, as well as its proven ability to overcome some common issues associated with immunoassay, namely selectivity and reagent availability. However, large proteins (>10 kDa) chromatograph and ionize poorly. To overcome this challenge, LC–MS/MS workflows for proteins larger than 10 kDa utilize enzymatic digestion procedures with subsequent quantitation of one or more of these enzymatically derived peptides to act as a surrogate for the intact protein. Here, recommendations of digestion technique and potential internal standards are summarized.