Quantitative Bottom-Up Proteomics Depends on Digestion Conditions

Abstract

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 cleanup, and denaturation temperature, among others. Timecourse experiments compared differences in relative peptide concentrations for tryptic digestions ranging from 15 min to 48 h. A predigested stable isotope-labeled ((15)N) form of the full-length (HSA) protein, expressed 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. Results demonstrate that 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 nonstoichiometry of enzymatic protein digestions emphasizes the often overlooked difficulties for routine absolute protein quantification, and highlights the need for use of suitable internal standards and isotope dilution techniques.

Transformative Impact of Proteomics on Cardiovascular Health and Disease

“…44 This highlights the need for standardized procedures to ensure accurate quantification of protein samples and suitable internal standards and isotope dilution techniques to improve uniformity of proteomic analysis. 44 At a minimum, full documentation of the digestion protocol is required so that other researchers can replicate the results and determine approach utility.…”

“…44 This highlights the need for standardized procedures to ensure accurate quantification of protein samples and suitable internal standards and isotope dilution techniques to improve uniformity of proteomic analysis. 44 At a minimum, full documentation of the digestion protocol is required so that other researchers can replicate the results and determine approach utility. A protocol to increase the reproducibility and reliability of proteomic workflows has been proposed and involves the addition of 3 exogenous proteins and 2 sets of isotopically labeled peptides (1 added before tryptic digestion and the other before LC-MS analysis).…”

Tandem Mass Spectral Libraries of Peptides in Digests of Individual Proteins: Human Serum Albumin (HSA)

“…Many of these were generated for two studies examining digestion variability (26,27). These served to generate peptides over a wide range of conditions and HSA sources, including 12 HSA samples from five vendors, eight sources of trypsin, and a range of denaturing/digestion conditions.…”

Quantitative Performance of Internal Standard Platforms for Absolute Protein Quantification Using Multiple Reaction Monitoring-Mass Spectrometry

“…[18][19][20][21] Several conditions for protein digestion were evaluated for optimal peptide release including denaturant, pH, temperature, enzyme, and enzyme concentration ( Figure 1). Unfortunately, but not unexpectedly, a single set of digestion conditions optimal for all peptides was not found.…”