Understanding the Role of Proteolytic Digestion on Discovery - and Targeted-Proteome Measurements using Liquid Chromatography Tandem Mass Spectrometry and Design of Experiments

摘要

Responses in global proteomic experiments are heavily influenced by incubation temperature, pH, enzyme:substrate ratio as well as the type of trypsin used. Digestion parameters effect the limit of detection and our ability to accurately quantify during absolute quantification PC-IDMS experiments. An enzyme:substrate ratio sufficient for "complete digestion" is critical for accurate quantification. Time course experiments revealed chymotryptic peptide production and correlated this production with tryptic peptide decay under previous digest conditions while there was no significant observable tryptic peptide decay under optimal digest conditions. A significant proportion of the observed tryptic peptide decay can be accounted for by the quantified chymotryptic activity.