Proteomic and phosphoproteomic comparison of human ES and iPS cells

Journal name:
Nature Methods
Volume:
8,
Pages:
821–827
Year published:
DOI:
doi:10.1038/nmeth.1699
Received
Accepted
Published online

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.

At a glance

Figures

  1. Figures of merit for peptide identification and quantification.
    Figure 1: Figures of merit for peptide identification and quantification.

    (a) Peptide identifications as a function of precursor and product mass tolerance. Using proteins isolated from human ESC whole-cell lysate, we performed liquid chromatography tandem mass spectrometry for each combination of dissociation method and mass analyzer. IT, ion-trap detection; FT, orbitrap detection. We searched data using fragment-ion tolerances of 0.01–5.0 Da, filtered results by precursor mass tolerances of 0.5–1,000 p.p.m. and filtered identifications to achieve 1% FDR. We performed experiments in triplicate and averaged the results. The number of peptide spectrum matches (PSMs) is proportional to circle size; number of unique peptides is represented by circle color as indicated. (b) R2 values for all peptides in each protein (H1 versus NFF comparison; fourplex experiment) were calculated as a metric for quality of quantification. (c) Characterization of quantification. Data points represent reporter ion intensities for a single protein mixed in the indicated ratios. Lines represent the theoretical value for the mixtures presented.

  2. A transcriptomic, proteomic and phosphoproteomic comparison of ESC lines H1 and H9, iPSC line DF19. 7 and NFF line.
    Figure 2: A transcriptomic, proteomic and phosphoproteomic comparison of ESC lines H1 and H9, iPSC line DF19. 7 and NFF line.

    (a) Heat maps depict all quantified transcripts, proteins and phosphorylation sites. Values were median-normalized. (b) Overlap between transcripts and proteins identified in the fourplex experiment. We considered transcripts 'present' if the reads per kilobase of exon per million mapped reads (RPKM) value was greater than 1 for all four cell types, and we determined protein identification via P-value filtering (1% FDR). (c) Cytoscape schematic of mRNA, protein and phosphorylation quantification from the fourplex experiment for genes known to have an interaction with NANOG, SOX2 or POU5F1 (search tool for the retrieval of interacting genes-proteins (STRING) database, confidence score > 0.90). Data are identified by protein name.

  3. Kinase substrate analysis between ESCs and NFFs (adapted from ref. 24 with permission from the American Association for the Advancement of Science).
    Figure 3: Kinase substrate analysis between ESCs and NFFs (adapted from ref. 24 with permission from the American Association for the Advancement of Science).

    Highlighted are kinase substrates for sets of phosphorylation sites that were enriched (changed by more than twofold) in ESCs (red; P < 0.05) and in NFFs (blue; P < 0.05).

  4. Comparison of four ESC and four iPSC lines.
    Figure 4: Comparison of four ESC and four iPSC lines.

    (a) Differentially regulated transcripts, proteins and phosphorylation sites are shown as a function of the number of comparisons (n). We performed differential expression analysis using subsets of data. For example, the n = 2 value reflects the number of differences detected from comparing just two ESC lines and two iPSC lines without biological replicate, whereas n = 12 represents the differences detected from comparing all four ESC lines and all four iPSC lines in biological triplicate. The number of differentially regulated elements for a given fold difference is indicated by different colors. The lines connect data point for ease of interpretation. (b) Heatmaps depicting differentially regulated transcripts, proteins and phosphorylation sites (P < 0.05, Student's t-test, with Benjamini-Hochberg correction). Only transcripts exhibiting at least a 1.5-fold difference and protein and phosphorylation sites exhibiting at least a 1.2-fold difference are shown. (c) Randomly selected examples of differentially regulated transcripts, proteins and phosphorylation sites. Bar heights represent relative reporter ion intensity (arbitrary units). *P < 0.05 (Student's t-test), (ESCs compared to iPSCs). (d) Differentially regulated transcripts detected based on either a comparison between biological triplicates of H1 and DF4.7 cell lines or a comparison of biological triplicates of all four ESC and all four iPSC lines. (e) Overlap between differentially regulated proteins and transcripts (left; only genes with both a quantified protein and transcript were included) and differentially regulated proteins and phosphorylation sites (right; only genes with both a quantified protein and phosphorylation site were included).

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Author information

  1. These authors contributed equally to this work.

    • Douglas H Phanstiel &
    • Justin Brumbaugh

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

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.

Competing financial 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.

Corresponding author

Correspondence to:

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Supplementary information

PDF files

  1. Supplementary Text and Figures (10M)

    Supplementary Figures 1–5, Supplementary Tables 4,8,9

Excel files

  1. Supplementary Table 1 (6M)

    Proteomic identification and quantification.

  2. Supplementary Table 2 (10M)

    Phosphoproteomic identification and quantification.

  3. Supplementary Table 3 (717K)

    Enrichment analysis from fourplex experiment.

  4. Supplementary Table 5 (5M)

    Transcriptomic identification and quantification.

  5. Supplementary Table 6 (1M)

    Transcripts, proteins, and phosphorylation sites that differ between ESCs and iPSCs.

  6. Supplementary Table 7 (238K)

    Enrichment analysis from eightplex experiment.

Additional data