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Quantitative bottom-up proteomics depends on digestion conditions



Mark S. Lowenthal, Yuxue Liang, Karen W. Phinney, Stephen E. Stein


Accurate quantification is a fundamental requirement in the fields of proteomics and biomarker discovery, and for clinical diagnostic assays. To demonstrate the extent of quantitative variability in measurable peptide concentrations due to differences among ‘typical’ protein digestion protocols, the model protein, human serum albumin (HSA), was subjected to enzymatic digestion using 12 different sample preparation methods, and separately, was examined through a comprehensive timecourse of trypsinolysis. A variety of digestion conditions were explored including differences in digestion time, denaturant, source of enzyme, sample clean-up, and denaturation temperature, among others. Timecourse experiments compared differences in relative peptide concentrations for tryptic digestions ranging from 15 minutes to 48 hours. A pre-digested stable isotope-labeled (15N) form of the full-length (HSA) protein, expressed and purified in yeast was spiked into all samples prior to LC-MS analysis to compare yields of numerous varieties of tryptic peptides. Relative quantification was achieved by normalization of integrated extracted ion chromatograms (XICs) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) by multiple-reaction monitoring (MRM) on a triple quadrupole (QQQ) MS. Related peptide fragmentation transitions, and multiple peptide charge states, were monitored for validation of quantitative results. In general, protein concentration was shown to be unequal to tryptic peptide concentrations for most peptides, including so-called ‘proteotypic’ peptides. Peptide release during digestion displayed complex kinetics dependent on digestion conditions and, by inference, from denatured protein structure. Hydrolysis rates at tryptic cleavage sites were also shown to be affected by differences in nearest and next-nearest amino acid residues. The data suggesting non-stoichiometry of enzymatic protein digestions emphasizes the often overlooked difficulties for rou
Analytical Chemistry


enzyme digestion variability, trypsin, proteomics, quantification, mass spectrometry, albumin


Lowenthal, M. , Liang, Y. , Phinney, K. and Stein, S. (2013), Quantitative bottom-up proteomics depends on digestion conditions, Analytical Chemistry, [online], (Accessed May 21, 2024)


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Created December 2, 2013, Updated November 10, 2018