title: 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.
authors.affiliation: Bioanalytical Science & Toxicokinetics, Drug Metabolism & Pharmacokinetics, Platform Technology & Science, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Szapacs
authors.affiliation: Bioanalytical Science & Toxicokinetics, Drug Metabolism & Pharmacokinetics, Platform Technology & Science, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mencken
authors.affiliation: Chemical Sciences, Molecular Drug Research, Platform Technology & Science, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Jon
authors.initials: J
authors.lastname: Williams
authors.affiliation: Bioanalytical Science & Toxicokinetics, Drug Metabolism & Pharmacokinetics, Platform Technology & Science, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D004798:Enzymes / Q000378:metabolism*; D006801:Humans; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D059748:Proteolysis; D012015:Reference Standards
chemical_list: D004798:Enzymes; D011506:Proteins