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Proteomic and phosphoproteomic comparison of human ES and iPS cells

Nature Methods volume 8, pages 821–827 (2011)Cite this article

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Abstract

Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell–Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

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Figure 1: Figures of merit for peptide identification and quantification.
Figure 2: A transcriptomic, proteomic and phosphoproteomic comparison of ESC lines H1 and H9, iPSC line DF19. 7 and NFF line.
Figure 3: Kinase substrate analysis between ESCs and NFFs (adapted from ref. 24 with permission from the American Association for the Advancement of Science).
Figure 4: Comparison of four ESC and four iPSC lines.

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References

  1. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).

    Article  CAS  Google Scholar 

  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  Google Scholar 

  3. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  PubMed  Google Scholar 

  4. Chin, M.H. et al. Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell 5, 111–123 (2009).

    Article  CAS  Google Scholar 

  5. Guenther, M.G. et al. Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. Cell Stem Cell 7, 249–257 (2010).

    Article  CAS  Google Scholar 

  6. Chin, M.H., Pellegrini, M., Plath, K. & Lowry, W.E. Molecular analyses of human induced pluripotent stem cells and embryonic stem cells. Cell Stem Cell 7, 263–269 (2010).

    Article  CAS  Google Scholar 

  7. Bock, C. et al. Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144, 439–452 (2011).

    Article  CAS  Google Scholar 

  8. Stadtfeld, M. et al. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465, 175–181 (2010).

    Article  CAS  Google Scholar 

  9. Doi, A. et al. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat. Genet. 41, 1350–1353 (2009).

    Article  CAS  Google Scholar 

  10. Lister, R. et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68–73 (2011).

    Article  CAS  Google Scholar 

  11. Olsen, J.V. et al. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 4, 709–712 (2007).

    Article  CAS  Google Scholar 

  12. McAlister, G.C., Phanstiel, D., Wenger, C.D., Lee, M.V. & Coon, J.J. Analysis of tandem mass spectra by FTMS for improved large-scale proteomics with superior protein quantification. Anal. Chem. 82, 316–322 (2010).

    Article  CAS  Google Scholar 

  13. Olsen, J.V. et al. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed. Mol. Cell. Proteomics 8, 2759–2769 (2009).

    Article  CAS  Google Scholar 

  14. Nagaraj, N., D′Souza, R.C.J., Cox, J., Olsen, J.V. & Mann, M. Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation. J. Proteome Res. 9, 6786–6794 (2010).

    Article  CAS  Google Scholar 

  15. Thompson, A. et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003).

    Article  CAS  Google Scholar 

  16. Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    Article  CAS  Google Scholar 

  17. Choe, L. et al. 8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease. Proteomics 7, 3651–3660 (2007).

    Article  CAS  Google Scholar 

  18. Ow, S.Y. et al. iTRAQ underestimation in simple and complex mixtures: the good, the bad and the ugly. J. Proteome Res. 8, 5347–5355 (2009).

    Article  CAS  Google Scholar 

  19. Wenger, C.D., Phanstiel, D.H., Lee, M.V., Bailey, D.J. & Coon, J.J. COMPASS: a suite of pre- and post-search proteomics software tools for OMSSA. Proteomics 6, 1064–1074 (2011).

    Article  Google Scholar 

  20. Shadforth, I.P., Dunkley, T.P.J., Lilley, K.S. & Bessant, C. i-Tracker: for quantitative proteomics using iTRAQ (TM). BMC Genomics 6, 145 (2005).

    Article  Google Scholar 

  21. Griffin, T.J. et al. iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. J. Proteome Res. 6, 4200–4209 (2007).

    Article  CAS  Google Scholar 

  22. Becker, K.A., Stein, J.L., Lian, J.B., van Wijnen, A.J. & Stein, G.S. Establishment of histone gene regulation and cell cycle checkpoint control in human embryonic stem cells. J. Cell. Physiol. 210, 517–526 (2007).

    Article  CAS  Google Scholar 

  23. Xue, Y. et al. GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Mol. Cell. Proteomics 7, 1598–1608 (2008).

    Article  CAS  Google Scholar 

  24. Manning, G., Whyte, D.B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

    Article  CAS  Google Scholar 

  25. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57, 289–300 (1995).

    Google Scholar 

  26. Ashburner, M. et al. Gene Ontology: tool for the unification of biology. Nat. Genet. 25, 25–29 (2000).

    Article  CAS  Google Scholar 

  27. Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30 (2000).

    Article  CAS  Google Scholar 

  28. Frye, M. & Watt, F.M. The RNA methyltransferase Misu (NSun2) mediates Myc-induced proliferation and is upregulated in tumors. Curr. Biol. 16, 971–981 (2006).

    Article  CAS  Google Scholar 

  29. Singh, M.K. et al. The T-box transcription factor Tbx15 is required for skeletal development. Mech. Dev. 122, 131–144 (2005).

    Article  CAS  Google Scholar 

  30. Dong, F. et al. Pitx2 promotes development of splanchnic mesoderm-derived branchiomeric muscle. Development 133, 4891–4899 (2006).

    Article  CAS  Google Scholar 

  31. Kim, K. et al. Epigenetic memory in induced pluripotent stem cells. Nature 467, 285–290 (2010).

    Article  CAS  Google Scholar 

  32. Polo, J.M. et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat. Biotechnol. 28, 848–855 (2010).

    Article  CAS  Google Scholar 

  33. Hu, B.Y. et al. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc. Natl. Acad. Sci. USA 107, 4335–4340 (2010).

    Article  CAS  Google Scholar 

  34. Siu, I.M. et al. Coexpression of neuronatin splice forms promotes medulloblastoma growth. Neuro-oncol. 10, 716–724 (2008).

    Article  CAS  Google Scholar 

  35. Hargrave, M. et al. Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction. Dev. Dyn. 210, 79–86 (1997).

    Article  CAS  Google Scholar 

  36. Kawano, Y. et al. CRMP-2 is involved in kinesin-1-dependent transport of the Sra-1/WAVE1 complex and axon formation. Mol. Cell. Biol. 25, 9920–9935 (2005).

    Article  CAS  Google Scholar 

  37. Zhu, H., Coppinger, J.A., Jang, C.Y., Yates, J.R. III & Fang, G. FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J. Cell Biol. 183, 835–848 (2008).

    Article  CAS  Google Scholar 

  38. Bourke, E., Brown, J.A.L., Takeda, S., Hochegger, H. & Morrison, C.G. DNA damage induces Chk1-dependent threonine-160 phosphorylation and activation of Cdk2. Oncogene 29, 616–624 (2010).

    Article  CAS  Google Scholar 

  39. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).

    Article  CAS  Google Scholar 

  40. Sengupta, S. et al. Highly consistent, fully representative mRNA-Seq libraries from ten nanograms of total RNA. Biotechniques 49, 898–904 (2010).

    Article  CAS  Google Scholar 

  41. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10 R25 (2009).

  42. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  Google Scholar 

  43. Good, D.M. et al. Post-acquisition ETD spectral processing for increased peptide identifications. J. Am. Soc. Mass Spectrom. 20, 1435–1440 (2009).

    Article  CAS  Google Scholar 

  44. Geer, L.Y. et al. Open mass spectrometry search algorithm. J. Proteome Res. 3, 958–964 (2004).

    Article  CAS  Google Scholar 

  45. Kersey, P.J. et al. The International Protein Index: An integrated database for proteomics experiments. Proteomics 4, 1985–1988 (2004).

    Article  CAS  Google Scholar 

  46. Nesvizhskii, A.I. & Aebersold, R. Interpretation of shotgun proteomic data - The protein inference problem. Mol. Cell. Proteomics 4, 1419–1440 (2005).

    Article  CAS  Google Scholar 

  47. Swaney, D.L., Wenger, C.D., Thomson, J.A. & Coon, J.J. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 106, 995–1000 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge A.J. Bureta for helping with and providing illustrations, A. Williams and K. Eastman for editing the text, G.McAlister for assistance with instrumentation, J. Yu for providing transcriptomic data before publication and Thomson lab members for critical reading and discussion of this manuscript. This work was supported by the University of Wisconsin, the Beckman Foundation and US National Institutes of Health (NIH) grants R01GM080148 (to J.J.C.) and P01GM081629 (to J.A.T. and J.J.C.). D.H.P. acknowledges support from a NIH predoctoral traineeship, the Genomic Sciences Training Program, NIH grant 5T32HG002760.

Author information

Author notes

  1. Douglas H Phanstiel and Justin Brumbaugh: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA

    Douglas H Phanstiel, Craig D Wenger, Derek J Bailey, Danielle L Swaney, Mark A Tervo & Joshua J Coon

  2. Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA

    Douglas H Phanstiel, Justin Brumbaugh, Craig D Wenger, Derek J Bailey, Danielle L Swaney, Mark A Tervo & Joshua J Coon

  3. Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA

    Justin Brumbaugh & Joshua J Coon

  4. Morgridge Institute for Research, Madison, Wisconsin, USA

    Justin Brumbaugh, Shulan Tian, Mitchell D Probasco, Jennifer M Bolin, Victor Ruotti, Ron Stewart & James A Thomson

  5. Department of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin, USA

    James A Thomson

  6. Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California, USA

    James A Thomson

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Contributions

D.H.P. designed research, prepared samples, performed mass spectrometry, wrote software, analyzed data and wrote the manuscript. J.B. designed research, grew cells, prepared samples, analyzed data and wrote the manuscript. C.D.W. wrote software. S.T. and V.R. analyzed data. M.D.P. grew cells. D.J.B. designed websites. D.L.S. helped with phosphorylation analysis. M.A.T. optimized the labeling procedure. J.M.B. performed RNA sequencing. R.S. designed research and analyzed data. J.A.T. and J.J.C. designed research and wrote the manuscript.

Corresponding author

Correspondence to Joshua J Coon.

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Competing interests

J.A.T. is a founder, stockowner, consultant and board member of Cellular Dynamics International (CDI), and serves as scientific advisor to and has financial interests in Tactics II Stem Cell Ventures. J.J.C. is a consultant for Thermo Fisher Scientific.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 4,8,9 (PDF 9330 kb)

Supplementary Table 1

Proteomic identification and quantification. (XLSX 5966 kb)

Supplementary Table 2

Phosphoproteomic identification and quantification. (XLSX 9623 kb)

Supplementary Table 3

Enrichment analysis from fourplex experiment. (XLSX 696 kb)

Supplementary Table 5

Transcriptomic identification and quantification. (XLSX 5051 kb)

Supplementary Table 6

Transcripts, proteins, and phosphorylation sites that differ between ESCs and iPSCs. (XLSX 1265 kb)

Supplementary Table 7

Enrichment analysis from eightplex experiment. (XLSX 231 kb)

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Phanstiel, D., Brumbaugh, J., Wenger, C. et al. Proteomic and phosphoproteomic comparison of human ES and iPS cells. Nat Methods 8, 821–827 (2011). https://doi.org/10.1038/nmeth.1699

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