Abstract
We report a mass spectrometry–based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fnmeth.2518/MediaObjects/41592_2013_Article_BFnmeth2518_Fig1_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fnmeth.2518/MediaObjects/41592_2013_Article_BFnmeth2518_Fig2_HTML.jpg)
Similar content being viewed by others
References
Olsen, J.V. et al. Sci. Signal. 3, ra3 (2010).
Kim, W. et al. Mol. Cell 44, 325–340 (2011).
Udeshi, N.D. et al. Mol. Cell Proteomics 12, 825–831 (2013).
Choudhary, C. et al. Science 325, 834–840 (2009).
Ficarro, S.B. et al. Anal. Chem. 81, 4566–4575 (2009).
Pinkse, M.W., Lemeer, S. & Heck, A.J. Methods Mol. Biol. 753, 215–228 (2011).
Kim, S.C. et al. Mol. Cell 23, 607–618 (2006).
Xu, G., Paige, J.S. & Jaffrey, S.R. Nat. Biotechnol. 28, 868–873 (2010).
Ong, S.E. et al. Mol. Cell Proteomics 1, 376–386 (2002).
Mertins, P. et al. Mol. Cell Proteomics 11, M111.014423 (2012).
Villén, J. & Gygi, S.P. Nat. Protoc. 3, 1630–1638 (2008).
McNulty, D.E. & Annan, R.S. Mol. Cell Proteomics 7, 971–980 (2008).
Wang, Y. et al. Proteomics 11, 2019–2026 (2011).
Udeshi, N.D. et al. Mol. Cell Proteomics 11, 148–159 (2012).
Schwartz, D. & Gygi, S.P. Nat. Biotechnol. 23, 1391–1398 (2005).
Hunter, T. Mol. Cell 28, 730–738 (2007).
Cheng, S.W. et al. Mol. Cell Biol. 32, 4691–4704 (2012).
Yang, F., Shen, Y., Camp, D.G. II. & Smith, R.D. Expert Rev. Proteomics 9, 129–134 (2012).
Rappsilber, J., Mann, M. & Ishihama, Y. Nat. Protoc. 2, 1896–1906 (2007).
Cox, J. et al. J. Proteome Res. 10, 1794–1805 (2011).
Acknowledgements
This work was supported in part by the Broad Institute of MIT and Harvard and by the following grants from the US National Institutes of Health: grant U24CA160034 from the National Cancer Institute Clinical Proteomics Tumor Analysis Consortium Initiative (to S.A.C.) and grants HHSN268201000033C and R01HL096738 from the National Heart, Lung, and Blood Institute (to S.A.C.).
Author information
Authors and Affiliations
Contributions
P.M. and S.A.C. developed the SEPTM strategy and conceived of the study; P.M., J.W.Q. and J.P. performed experiments; P.M., J.W.Q., J.P., N.D.U., K.R.C., D.R.M., M.W.B., M.A.G., J.D.J. and S.A.C. contributed to experimental design and data analysis; and P.M. and S.A.C. wrote the manuscript with input from all authors.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Combined PDF
Supplementary Figures 1–4 and Supplementary Tables 5–7 (PDF 747 kb)
Supplementary Table 1
List of all quantified proteins, pSTY-, K(GG)- and K(Ac)-sites that were quantified in at least two replicates in the high, medium and low coverage experiments. (XLSX 22886 kb)
Supplementary Table 2
List of all quantified peptides observed in the proteome analysis at high, medium and low coverage of one representative replicate (replicate 1). (XLSX 28756 kb)
Supplementary Table 3
List of all quantified peptides observed in the phosphoproteome, ubiquitinome and acetylome analyses at high, medium and low coverage of one representative replicate (replicate 1). (XLSX 22298 kb)
Supplementary Table 4
Ubiquitin- and acetyl-site tables for label-free and SILAC-based comparison of non-serial and serial enrichments. (XLSX 2662 kb)
Rights and permissions
About this article
Cite this article
Mertins, P., Qiao, J., Patel, J. et al. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10, 634–637 (2013). https://doi.org/10.1038/nmeth.2518
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.2518
This article is cited by
-
IKAROS: from chromatin organization to transcriptional elongation control
Cell Death & Differentiation (2023)
-
c-Myc uses Cul4b to preserve genome integrity and promote antiviral CD8+ T cell immunity
Nature Communications (2023)
-
The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis
Communications Biology (2022)
-
Mitochondrial Fus1/Tusc2 and cellular Ca2+ homeostasis: tumor suppressor, anti-inflammatory and anti-aging implications
Cancer Gene Therapy (2022)
-
Ubiquitination of NF-κB p65 by FBXW2 suppresses breast cancer stemness, tumorigenesis, and paclitaxel resistance
Cell Death & Differentiation (2022)