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Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Proteome Res. 2014, 13, 9, 3979-3986
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Influence of the Digestion Technique, Protease, and Missed Cleavage Peptides in Protein Quantitation

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† Proteomics Unit, Centre de Regulació Genòmica (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain
‡ Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003 Barcelona, Spain
*Tel: +34 933 160 834. Fax: +34 933 160 099. E-mail: [email protected]
Cite this: J. Proteome Res. 2014, 13, 9, 3979–3986
Publication Date (Web):July 2, 2014
https://doi.org/10.1021/pr500294d
Copyright © 2014 American Chemical Society
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Abstract

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Quantitative determination of absolute and relative protein amounts is an essential requirement for most current bottom-up proteomics applications, but protein quantitation estimates are affected by several sources of variability such as sample preparation, mass spectrometric acquisition, and data analysis. Among them, sample digestion has attracted much attention from the proteomics community, as protein quantitation by bottom-up proteomics relies on the efficiency and reproducibility of protein enzymatic digestion, with the presence of missed cleavages, nonspecific cleavages, or even the use of different proteases having been postulated as important sources of variation in protein quantitation. Here we evaluated both in-solution and filter-aided digestion protocols and assessed their influence in the estimation of protein abundances using five E. coli mixtures with known amounts of spiked proteins. We observed that replicates of trypsin specificity digestion protocols are highly reproducible in terms of peptide quantitation, with digestion technique and the chosen proteolytic enzyme being the major sources of variability in peptide quantitation. Finally, we also evaluated the result of including peptides with missed cleavages in protein quantitation and observed no significant differences in precision, accuracy, specificity, and sensitivity compared with the use of fully tryptic peptides.

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Supplementary Table ST1. List of the SwissProt accession numbers divided by subsets corresponding to the controlled proteins spiked in an E. coli background. Supplementary Table ST2. Complete set of inputs and outputs from MSstats. Supplementary Table ST3. Summary table of the outputs from MSstats for the assessment of protein quantitation of different sample digestion protocols. Supplementary Table ST4. List of protein groups and peptides identified in each digestion protocol in the E. coli samples with spiked-in control proteins. Supplementary Table ST5. Pearson and Spearman correlation values of the extracted peptide areas corresponding to Figures 2A,B and 4B. Supplementary Table ST6. List of protein groups and peptides identified in each digestion protocol in the HeLa sample. Supplementary Figure S1. Sensitivity and specificity of relative protein quantitation for all different values of protein fold-changes calculated with all peptides or only including peptides without any missed cleavages. Sensitivity was calculated as the proportion of proteins known to change that had a significant p value. Similarly, specificity referred to the proportion of nonchanging proteins that were correctly identified as such (i.e., having a nonsignificant p value). Supplementary Figure S2. Distribution of median normalized peptide log 2 intensities from different digestion protocols represented by a boxplot. This material is available free of charge via the Internet at http://pubs.acs.org.

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