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Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells

Abstract

Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R2 = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R2 = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.

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Figure 1: Validation of improvements incorporated in the iST method.
Figure 2: Minimal sample-processing protocol performed in an enclosed volume is amenable to automation and scaling.
Figure 3: Quantitative reproducibility of in-depth analysis of S. cerevisiae proteome and copy-number estimation.
Figure 4: In-depth analysis of yeast proteomes and estimation of yeast copy numbers.
Figure 5: In-depth analysis of the human proteome and estimation of copy numbers using the iST method.

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References

  1. Altelaar, A.M. & Heck, A.J. Trends in ultrasensitive proteomics. Curr. Opin. Chem. Biol. 16, 206–213 (2012).

    Article  CAS  Google Scholar 

  2. de Godoy, L.M. et al. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 455, 1251–1254 (2008).

    Article  CAS  Google Scholar 

  3. Thakur, S.S. et al. Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell. Proteomics 10, M110 003699 (2011).

    Article  Google Scholar 

  4. Kocher, T., Swart, R. & Mechtler, K. Ultra-high-pressure RPLC hyphenated to an LTQ-Orbitrap Velos reveals a linear relation between peak capacity and number of identified peptides. Anal. Chem. 83, 2699–2704 (2011).

    Article  Google Scholar 

  5. Nagaraj, N. et al. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap. Mol. Cell. Proteomics 11, M111 013722 (2012).

    Article  Google Scholar 

  6. Yamana, R. et al. Rapid and deep profiling of human induced pluripotent stem cell proteome by one-shot NanoLC-MS/MS analysis with meter-scale monolithic silica columns. J. Proteome Res. 12, 214–221 (2013).

    Article  CAS  Google Scholar 

  7. Michalski, A. et al. Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol. Cell. Proteomics 10, M111 011015 (2011).

    Article  Google Scholar 

  8. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    Article  CAS  Google Scholar 

  9. Chen, E.I., McClatchy, D., Park, S.K. & Yates, J.R. III. Comparisons of mass spectrometry compatible surfactants for global analysis of the mammalian brain proteome. Anal. Chem. 80, 8694–8701 (2008).

    Article  CAS  Google Scholar 

  10. Nagaraj, N., Lu, A., Mann, M. & Wisniewski, J.R. Detergent-based but gel-free method allows identification of several hundred membrane proteins in single LC-MS runs. J. Proteome Res. 7, 5028–5032 (2008).

    Article  CAS  Google Scholar 

  11. Manza, L.L., Stamer, S.L., Ham, A.J., Codreanu, S.G. & Liebler, D.C. Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 5, 1742–1745 (2005).

    Article  CAS  Google Scholar 

  12. Wisniewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Article  CAS  Google Scholar 

  13. Ethier, M., Hou, W., Duewel, H.S. & Figeys, D. The proteomic reactor: a microfluidic device for processing minute amounts of protein prior to mass spectrometry analysis. J. Proteome Res. 5, 2754–2759 (2006).

    Article  CAS  Google Scholar 

  14. Zhou, H., Ning, Z., Wang, F., Seebun, D. & Figeys, D. Proteomic reactors and their applications in biology. FEBS J. 278, 3796–3806 (2011).

    Article  CAS  Google Scholar 

  15. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    Article  CAS  Google Scholar 

  16. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  17. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  18. Cox, J. & Mann, M. 1D and 2D annotation enrichment: a statistical method integrating quantitative proteomics with complementary high-throughput data. BMC Bioinformatics 13, S12 (2012).

    Article  CAS  Google Scholar 

  19. Poulsen, J.W., Madsen, C.T., Young, C., Poulsen, F.M. & Nielsen, M.L. Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry. J. Proteome Res. 12, 1020–1030 (2013).

    Article  CAS  Google Scholar 

  20. Leon, I.R., Schwammle, V., Jensen, O.N. & Sprenger, R.R. Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol. Cell. Proteomics 12, 2992–3005 (2013).

    Article  CAS  Google Scholar 

  21. Peng, M. et al. Protease bias in absolute protein quantitation. Nat. Methods 9, 524–525 (2012).

    Article  CAS  Google Scholar 

  22. Wisniewski, J.R. et al. Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma. Mol. Syst. Biol. 8, 611 (2012).

    Article  Google Scholar 

  23. Picotti, P., Bodenmiller, B., Mueller, L.N., Domon, B. & Aebersold, R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138, 795–806 (2009).

    Article  CAS  Google Scholar 

  24. Nieduszynski, C.A., Hiraga, S., Ak, P., Benham, C.J. & Donaldson, A.D. OriDB: a DNA replication origin database. Nucleic Acids Res. 35, D40–D46 (2007).

    Article  CAS  Google Scholar 

  25. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    Article  CAS  Google Scholar 

  26. Gunaratne, J. et al. Extensive mass spectrometry-based analysis of the fission yeast proteome: The S. pombe PeptideAtlas. Mol. Cell. Proteomics 12, 1741–1751 (2013).

    Article  CAS  Google Scholar 

  27. Marguerat, S. et al. Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells. Cell 151, 671–683 (2012).

    Article  CAS  Google Scholar 

  28. Powell, S. et al. eggNOG v3.0: orthologous groups covering 1133 organisms at 41 different taxonomic ranges. Nucleic Acids Res. 40, D284–D289 (2012).

    Article  CAS  Google Scholar 

  29. Zeiler, M., Straube, W.L., Lundberg, E., Uhlen, M. & Mann, M. A protein epitope signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines. Mol. Cell. Proteomics 11, O111.009613 (2012).

    Article  Google Scholar 

  30. Nagaraj, N. et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548 (2011).

    Article  Google Scholar 

  31. Beck, M. et al. The quantitative proteome of a human cell line. Mol. Syst. Biol. 7, 549 (2011).

    Article  Google Scholar 

  32. Schwanhausser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

    Article  Google Scholar 

  33. Cristobal, I. et al. PP2A impaired activity is a common event in acute myeloid leukemia and its activation by forskolin has a potent anti-leukemic effect. Leukemia 25, 606–614 (2011).

    Article  CAS  Google Scholar 

  34. Kar, R., Singha, P.K., Venkatachalam, M.A. & Saikumar, P. A novel role for MAP1 LC3 in nonautophagic cytoplasmic vacuolation death of cancer cells. Oncogene 28, 2556–2568 (2009).

    Article  CAS  Google Scholar 

  35. Tanida, I., Minematsu-Ikeguchi, N., Ueno, T. & Kominami, E. Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1, 84–91 (2005).

    Article  CAS  Google Scholar 

  36. Forsburg, S.L. Eukaryotic MCM proteins: beyond replication initiation. Microbiol. Mol. Biol. Rev. 68, 109–131 (2004).

    Article  CAS  Google Scholar 

  37. Williams, R.S., Williams, J.S. & Tainer, J.A. Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem. Cell Biol. 85, 509–520 (2007).

    Article  CAS  Google Scholar 

  38. Levin, J.Z. et al. Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat. Methods 7, 709–715 (2010).

    Article  CAS  Google Scholar 

  39. Schaab, C., Geiger, T., Stoehr, G., Cox, J. & Mann, M. Analysis of high accuracy, quantitative proteomics data in the MaxQB database. Mol. Cell. Proteomics 11, M111 014068 (2012).

    Article  Google Scholar 

  40. Masuda, T., Tomita, M. & Ishihama, Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 7, 731–740 (2008).

    Article  CAS  Google Scholar 

  41. Scheltema, R.A. & Mann, M. SprayQc: a real-time LC-MS/MS quality monitoring system to maximize uptime using off the shelf components. J. Proteome Res. (11 May 2012).

  42. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Braun and P. Georgescu (Ludwig Maximilian University of Munich) for providing us with fission yeast pellets, F. Sacco, M. Steger, D. Walther and M. Räschle for help concerning biological interpretations. M. Hein, A. Dalfovo, C. Schaab and J. Cox helped with figures, videos and bioinformatics analysis of our data sets. Work in M.M.'s laboratory is supported by the Max Planck Society for the Advancement of Science and PROSPECT, a 7th framework program of the European Union (grant agreement HEALTH-F4-2008-201648).

Author information

Authors and Affiliations

Authors

Contributions

N.A.K. and M.M. developed and invented the method; G.P. and N.N. contributed in the developments; N.A.K., G.P., I.P. and N.N. performed the experiments; and N.A.K., G.P., N.N. and M.M. designed and interpreted the experiments, and wrote the manuscript.

Corresponding author

Correspondence to Matthias Mann.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Note (PDF 1672 kb)

Supplementary Table 1

Protein identifications and protein copy number estimations of S.cerevisiae, S.pombe and HeLa cells. (XLSX 1707 kb)

Supplementary Table 2

Orthologs of S.cerevisiae, S.pombe and HeLa cells and their protein copy number estimations. (XLSX 931 kb)

Supplementary Table 3

Buffer compositions and materials for the iST method. (XLSX 12 kb)

iST sample-preparation video tutorial.

The video tutorial shows all steps of the iST sample preparation workflow. It also shows how to troubleshoot the method, if necessary. (MP4 27589 kb)

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Kulak, N., Pichler, G., Paron, I. et al. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11, 319–324 (2014). https://doi.org/10.1038/nmeth.2834

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