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Comparative Study
. 2013 Oct;12(10):2992-3005.
doi: 10.1074/mcp.M112.025585. Epub 2013 Jun 21.

Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis

Ileana R León  1 , Veit Schwämmle, Ole N Jensen, Richard R Sprenger
Affiliations
Comparative Study

Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis

VSports手机版 - Ileana R León et al. Mol Cell Proteomics. 2013 Oct.

Abstract

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods VSports手机版. .

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Figures

Fig. 1.
Fig. 1.
Study design. Flowchart illustrating the combination of different steps and digestion conditions compared in this study.
Fig. 2.
Fig. 2.
Qualitative comparison of seven digestion protocols analyzed with standard nanoLC-MS/MS. Protein (A) and peptide (C) identifications are reported as the merged result of three replicates per protocol. Depicted are the distributions of summed protein sequence coverage (B) and the percentage of missed cleavages among the evaluated protocols (D).
Fig. 3.
Fig. 3.
Qualitative evaluation of six digestion protocols analyzed with data independent LC-MSE. Average number of unique protein (A) and peptide (C) identifications from technical triplicates (white bars), number of quantified proteins and peptides present in at least two out of three replicates (gray bars), and average number of unique, quantified protein and peptide identifications after alignment of all data-independent runs using Progenesis LC-MS software (black bars). The distributions of summed protein sequence coverage are depicted (B) as well as the percentage of missed cleavages among the evaluated protocols (D).
Fig. 4.
Fig. 4.
Dynamic range distribution and stoichiometry of quantified mitochondrial proteins. The plotted abundance distribution (A) depicts results from the ISD:SDC (PT) protocol. Light blue circles represent proteins without transmembrane domains (TM = 0), whereas dark blue circles represent proteins containing one or more transmembrane domains (TM ≥ 1). Yellow diamonds represent quantified ATP synthase subunits, which agree with their known stoichiometry. The ATPsynthase complex is schematically depicted in the top right corner (A). The bar charts represent the summed absolute (B) and relative (C) protein amounts obtained for each protocol categorized by proteins with (TM ≥ 1) or without (TM = 0) transmembrane domains. (D) The detection and absolute amount of the catalytic core F1 complex subunits α3 β3 γ1 δ1 and ε1 for each of the investigated protocols. The error bars indicate standard deviation of triplicate measurements.
Fig. 5.
Fig. 5.
Quantitative evaluation of the relative protein and peptide abundance bias for each digestion protocol. Histograms represent the peptide (A) and protein (B) distribution among the generated bins of the log2(ratio). This ratio was calculated as the change in abundance for each peptide or protein in each protocol relative to the average of all digestion protocols. The interval among the dashed lines represents no significant change in peptide or protein abundances, which is defined as ± one standard deviation, calculated over the complete quantitative data set. The percentages above each histogram represent the peptides or proteins that are included in this interval. Box plot distribution of percentage ‘deviation from average’ for peptides (C) and proteins (D). The deviation from average is defined as the relative deviation of each peptide or protein in a particular protocol from the mean abundance of all protocols.
Fig. 6.
Fig. 6.
In-depth quantitative analysis of significant differences observed among protocols. A, D, Hierarchical clustering of peptide (A) and protein (D) normalized log ratios among each digestion protocol, measured in triplicate. The heatmap color limits are set to ± two standard deviations, calculated over the complete quantitative data set. B, E, Fuzzy c-means clustering of changes in peptide (B) and protein (E) standardized abundance among all protocols, presented in the same order as listed for the heatmaps. C, F, Summary of differences observed among each cluster for several physicochemical peptide (C) and protein (F) parameters, including peptide sequence length, pI, hydrophobicity (GRAVY), protein molecular weight (MW) and number of transmembrane (TM) domains. G, Bar graphs representing the summed log ratio of significantly changed protein and peptide abundances in each protocol plotted against several binned physicochemical parameters. Significance level was defined as q-value <0.05 (p value corrected for multiple testing) and ± one standard deviation calculated over the average of all protocols. This corresponds to a 1.5-fold and 2.2-fold change on peptide and protein level, respectively.
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