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Protease bias in absolute protein quantitation

Nature Methods volume 9, pages 524–525 (2012)Cite this article

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To the Editor:

Reliable ways to determine absolute protein abundances at a proteome-wide scale are in high demand. Label-free mass spectrometric approaches may fulfill this need because they are cost efficient, require little sample pre-treatment and can be used on any biological sample. In such approaches the spectral counts and/or intensity of the peptides detected in liquid chromatography–tandem mass spectrometry analyses correlate with the overall amount of the peptides' source protein from which the peptides originated1,2,3,4. Trypsin is by far the most popular enzyme used in proteomics, and huge efforts are currently underway to define spectral atlases of so-called proteotypic (tryptic) peptides for quantitative assays5. A potential drawback of proteases is their irreproducibility in peptide formation. Trypsin in particular has limited functionality under harsher digestion conditions. This may introduce a bias in the determination of absolute protein abundance6. To evaluate the extent of such a bias, we generated 14 comprehensive7, label-free quantitative proteomics data sets on Saccharomyces cerevisiae based on both spectral counts and ion volume (iBAQ4). We used four proteases (trypsin, chymotrypsin, Lys-C and Lys-N) and two instrument platforms (Supplementary Fig. 1, Supplementary Tables 1–3 and Supplementary Methods).

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Figure 1: Comparison of absolute protein quantification by spectral counts with different enzymes.

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Acknowledgements

This work was supported by the PRIME-XS project, grant agreement 262067, funded by the European Union 7th Framework Programme; the Netherlands Proteomics Centre of the Netherlands Genomics Initiative; the Centre for Biomedical Genetics; the Netherlands Organization for Scientific Research (NWO) VIDI grant (700.10.429) and the Netherlands Bioinformatics Centre.

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Authors and Affiliations

  1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Mao Peng, Nadia Taouatas, Salvatore Cappadona, Bas van Breukelen, Shabaz Mohammed, Arjen Scholten & Albert J R Heck

  2. Netherlands Proteomics Centre, Utrecht, The Netherlands

    Mao Peng, Nadia Taouatas, Salvatore Cappadona, Bas van Breukelen, Shabaz Mohammed, Arjen Scholten & Albert J R Heck

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  1. Mao Peng
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  2. Nadia Taouatas
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  3. Salvatore Cappadona
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Correspondence to Arjen Scholten or Albert J R Heck.

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Supplementary information

Supplementary Text and Figures

Supplementary Figure 1-3, Supplementary Table 4 and Supplementary Methods (PDF 1333 kb)

Supplementary Table

Supplementary Table 1 (XLSX 2336 kb)

Supplementary Table

Supplementary Table 2 (XLSX 3154 kb)

Supplementary Table

Supplementary Table 3 (XLSX 1338 kb)

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Peng, M., Taouatas, N., Cappadona, S. et al. Protease bias in absolute protein quantitation. Nat Methods 9, 524–525 (2012). https://doi.org/10.1038/nmeth.2031

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