Abstract
For ease of measurement and accurate identification of proteins by mass spectrometry, protein targets are commonly cleaved into peptides. Protein digestion is a critical step in sample preparation, yielding peptides amenable to both chromatographic separation and mass spectrometric analysis. Trypsin is the most extensively used protease due to its high cleavage specificity; however, it can yield highly variable digestion profiles and is dependent on several factors including digestion buffer, denaturant, trypsin quality selected, and composition/complexity of the sample matrix. Historically, trypsin digestion protocols have relied on lengthy digestion times-which are unsuitable for many clinical applications-to ensure effective proteolysis. Here, we performed an iterative and comprehensive evaluation of digestion conditions for five structurally diverse proteins in plasma and serum: apolipoprotein A-1, retinol-binding protein 4, transthyretin, complement component 9 and C-reactive protein. Conditions were monitored for improvements in signal intensity, reproducibility of digestion profile, and rate of release of proteolytic peptides. This approach yielded an optimized digestion protocol for detection of all five proteins in a single workflow requiring a brief 20 min digestion, without the use of chemical denaturants or reduction/alkylation steps, and only 1 μl of plasma. It is our hope that this data can accelerate the development phase of targeted mass spectrometric protein assays by identifying practical approaches to accelerate and simplify digestion protocols for clinical applications and assist with the selection of tryptic peptides for protein quantitation.