Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Top-down characterization of endogenous protein complexes with native proteomics


Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein–protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Native proteomics implemented in untargeted mode.
Figure 2: A summary of results from performing native proteomics.
Figure 3: Direct readout of metal cofactors by native proteomics.
Figure 4: Detection of labile superoxide bound to mitochondrial superoxide dismutase.

Accession codes


Protein Data Bank


  1. 1

    Varambally, S. et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419, 624–629 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Alcorta, D.A. et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl. Acad. Sci. USA 93, 13742–13747 (1996).

    CAS  PubMed  Google Scholar 

  3. 3

    Dunaief, J.L. et al. The retinoblastoma protein and BRG1 form a complex and cooperate to induce cell cycle arrest. Cell 79, 119–130 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Hummon, A.B. et al. From the genome to the proteome: uncovering peptides in the Apis brain. Science 314, 647–649 (2006).

    CAS  PubMed  Google Scholar 

  5. 5

    Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 (2009).

    CAS  Google Scholar 

  6. 6

    Tran, J.C. et al. Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature 480, 254–258 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Catherman, A.D. et al. Large-scale top-down proteomics of the human proteome: membrane proteins, mitochondria, and senescence. Mol. Cell. Proteomics 12, 3465–3473 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Wu, S. et al. Top-down characterization of the post-translationally modified intact periplasmic proteome from the bacterium Novosphingobium aromaticivorans. Int. J. Proteomics 2013, 279590 (2013).

    PubMed  PubMed Central  Google Scholar 

  9. 9

    Zhou, M. et al. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334, 380–385 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Li, H., Wongkongkathep, P., Van Orden, S.L., Ogorzalek Loo, R.R. & Loo, J.A. Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment. J. Am. Soc. Mass Spectrom. 25, 2060–2068 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Fenn, J.B., Mann, M., Meng, C.K., Wong, S.F. & Whitehouse, C.M. Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64–71 (1989).

    CAS  PubMed  Google Scholar 

  12. 12

    Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Baradaran, R., Berrisford, J.M., Minhas, G.S. & Sazanov, L.A. Crystal structure of the entire respiratory complex I. Nature 494, 443–448 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Bachir, A.I. et al. Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions. Curr. Biol. 24, 1845–1853 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Du, J., Lü, W., Wu, S., Cheng, Y. & Gouaux, E. Glycine receptor mechanism elucidated by electron cryo-microscopy. Nature 526, 224–229 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Kühlbrandt, W. Biochemistry. The resolution revolution. Science 343, 1443–1444 (2014).

    PubMed  Google Scholar 

  17. 17

    Sheng, M., Cummings, J., Roldan, L.A., Jan, Y.N. & Jan, L.Y. Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368, 144–147 (1994).

    CAS  Google Scholar 

  18. 18

    Link, A.J. et al. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682 (1999).

    CAS  PubMed  Google Scholar 

  19. 19

    Havugimana, P.C. et al. A census of human soluble protein complexes. Cell 150, 1068–1081 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Huttlin, E.L. et al. The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell 162, 425–440 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Rees, J.S. et al. In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry. Mol. Cell Proteomics 10, M110.2386 (2011).

    Google Scholar 

  22. 22

    Belov, M.E. et al. From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry. Anal. Chem. 85, 11163–11173 (2013).

    CAS  Google Scholar 

  23. 23

    Skinner, O.S. et al. Native GELFrEE: a new separation technique for biomolecular assemblies. Anal. Chem. 87, 3032–3038 (2015).

    CAS  Google Scholar 

  24. 24

    Melani, R.D. et al. CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis. J. Vis. Exp. 2016, 53597 (2016).

    Google Scholar 

  25. 25

    Skinner, O.S. et al. An informatic framework for decoding protein complexes by top-down mass spectrometry. Nat. Methods 13, 237–240 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Smith, L.M., Kelleher, N.L. & The Consortium for Top Down Proteomics Proteoform: a single term describing protein complexity. Nat. Methods 10, 186–187 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Havugimana, P.C., Wong, P. & Emili, A. Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 847, 54–61 (2007).

    CAS  Google Scholar 

  28. 28

    Skinner, O.S., Schachner, L.F. & Kelleher, N.L. The search engine for multi-proteoform complexes: an online tool for the identification and stoichiometry determination of protein complexes. Curr. Protoc. Bioinformatics 56, 13.30.1–13.30.11 (2016).

    Google Scholar 

  29. 29

    van de Waterbeemd, M. et al. High-fidelity mass analysis unveils heterogeneity in intact ribosomal particles. Nat. Methods 14, 283–286 (2017).

    CAS  PubMed  Google Scholar 

  30. 30

    Bakkenist, C.J. & Kastan, M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

    CAS  PubMed  Google Scholar 

  31. 31

    McMahon, C.G. et al. Diagnostic accuracy of heart-type fatty acid-binding protein for the early diagnosis of acute myocardial infarction. Am. J. Emerg. Med. 30, 267–274 (2012).

    PubMed  Google Scholar 

  32. 32

    Liu, F., Rijkers, D.T., Post, H. & Heck, A.J. Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat. Methods 12, 1179–1184 (2015).

    CAS  PubMed  Google Scholar 

  33. 33

    Foster, A.W., Osman, D. & Robinson, N.J. Metal preferences and metallation. J. Biol. Chem. 289, 28095–28103 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Zheng, W. et al. The crystal structure of human isopentenyl diphosphate isomerase at 1.7 A resolution reveals its catalytic mechanism in isoprenoid biosynthesis. J. Mol. Biol. 366, 1447–1458 (2007).

    CAS  PubMed  Google Scholar 

  35. 35

    Costello, L.C., Liu, Y., Franklin, R.B. & Kennedy, M.C. Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cells. J. Biol. Chem. 272, 28875–28881 (1997).

    CAS  PubMed  Google Scholar 

  36. 36

    Robbins, A.H. & Stout, C.D. Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal. Proc. Natl. Acad. Sci. USA 86, 3639–3643 (1989).

    CAS  PubMed  Google Scholar 

  37. 37

    Kang, H.J., Jung, S.K., Kim, S.J. & Chung, S.J. Structure of human alpha-enolase (hENO1), a multifunctional glycolytic enzyme. Acta Crystallogr. D Biol. Crystallogr. 64, 651–657 (2008).

    CAS  PubMed  Google Scholar 

  38. 38

    Emanuelsson, O., Nielsen, H., Brunak, S. & von Heijne, G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 300, 1005–1016 (2000).

    CAS  Google Scholar 

  39. 39

    Oyer, J.A. et al. Point mutation E1099K in MMSET/NSD2 enhances its methyltranferase activity and leads to altered global chromatin methylation in lymphoid malignancies. Leukemia 28, 198–201 (2014).

    CAS  PubMed  Google Scholar 

  40. 40

    Durbin, K.R. et al. Quantitation and identification of thousands of human proteoforms below 30 kDa. J. Proteome Res. 15, 976–982 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Yang, Y., Barendregt, A., Kamerling, J.P. & Heck, A.J. Analyzing protein micro-heterogeneity in chicken ovalbumin by high-resolution native mass spectrometry exposes qualitatively and semi-quantitatively 59 proteoforms. Anal. Chem. 85, 12037–12045 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Sasaki, T. & Takai, Y. The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem. Biophys. Res. Commun. 245, 641–645 (1998).

    CAS  PubMed  Google Scholar 

  43. 43

    Eliot, A.C. & Kirsch, J.F. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Reddy, S., Jones, A.D., Cross, C.E., Wong, P.S. & Van Der Vliet, A. Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue. Biochem. J. 347, 821–827 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Wilson, M.A. The role of cysteine oxidation in DJ-1 function and dysfunction. Antioxid. Redox Signal. 15, 111–122 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Vigushin, D.M. et al. Trichostatin A is a histone deacetylase inhibitor with potent antitumor activity against breast cancer in vivo. Clin. Cancer Res. 7, 971–976 (2001).

    CAS  PubMed  Google Scholar 

  47. 47

    Zhao, S. et al. Regulation of cellular metabolism by protein lysine acetylation. Science 327, 1000–1004 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Weinert, B.T. et al. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. Mol. Syst. Biol. 10, 716 (2014).

    PubMed  PubMed Central  Google Scholar 

  49. 49

    Skinner, O.S. & Kelleher, N.L. Illuminating the dark matter of shotgun proteomics. Nat. Biotechnol. 33, 717–718 (2015).

    CAS  PubMed  Google Scholar 

  50. 50

    Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 81, 153–208 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Zhang, Z. & Marshall, A.G. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J. Am. Soc. Mass Spectrom. 9, 225–233 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Strohalm, M., Kavan, D., Novák, P., Volný, M. & Havlícek, V. mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal. Chem. 82, 4648–4651 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Ruepp, A. et al. CORUM: the comprehensive resource of mammalian protein complexes--2009. Nucleic Acids Res. 38, D497–D501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


Funding for this project was provided by the W.M. Keck foundation (DT061512) and Northwestern University. This research was also supported by the Paul G. Allen Family Foundation (Grant Award 11715). The authors would also like to acknowledge helpful discussion with A. Catherman, R. Fellers, J. Greer, and B. Early. Helpful assistance from Thermo Fisher Scientific was provided by M. Belov, S. Horning, and A. Makarov. OSS and PFD are supported by US National Science Foundation graduate research fellowships (2014171659 and 2015210477, respectively). LHFDV is supported under CNPq research grant 400301/2014-8 from the Brazilian government. LFS was supported by the Chemistry of Life Processes Predoctoral training program at Northwestern University. Additional support for the maintenance of the SEMPC from the National Resource for Translational and Developmental Proteomics (GM108569) is acknowledged.

Author information




Data acquisition and analysis was performed by O.S.S. with assistance from N.A.H., L.F., R.D.M., and from all authors. N.A.H., R.D.M., L.F., L.H.F.D.V., H.S.S., and P.F.D. prepared samples with help from O.S.S. and L.F.S. O.S.S. wrote the manuscript with critical insights from P.D.C. and additional assistance from K.S. and all co-authors. N.L.K. and P.D.C. conceived of and oversaw the project.

Corresponding authors

Correspondence to Neil L Kelleher or Philip D Compton.

Ethics declarations

Competing interests

N.L.K. serves as a paid consultant to Thermo Fisher Scientific, whose instruments were used in this work.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–18 (PDF 25511 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Supplementary Dataset 1

Supplementary Data Set 1 (XLSX 26 kb)

Supplementary Dataset 2

Supplementary Data Set 2 (XLSX 36 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Skinner, O., Haverland, N., Fornelli, L. et al. Top-down characterization of endogenous protein complexes with native proteomics. Nat Chem Biol 14, 36–41 (2018).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing