The Search Engine for Multi‐Proteoform Complexes: An Online Tool for the Identification and Stoichiometry Determination of Protein Complexes

Owen S. Skinner1, Luis F. Schachner1, Neil L. Kelleher2

1 Department of Chemistry, Northwestern University, Evanston, Illinois, 2 Department of Molecular Biosciences, Northwestern University, Evanston, Illinois
Publication Name:  Current Protocols in Bioinformatics
Unit Number:  Unit 13.30
DOI:  10.1002/cpbi.16
Online Posting Date:  December, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Recent advances in top‐down mass spectrometry using native electrospray now enable the analysis of intact protein complexes with relatively small sample amounts in an untargeted mode. Here, we describe how to characterize both homo‐ and heteropolymeric complexes with high molecular specificity using input data produced by tandem mass spectrometry of whole protein assemblies. The tool described is a “search engine for multi‐proteoform complexes,” (SEMPC) and is available for free online. The output is a list of candidate multi‐proteoform complexes and scoring metrics, which are used to define a distinct set of one or more unique protein subunits, their overall stoichiometry in the intact complex, and their pre‐ and post‐translational modifications. Thus, we present an approach for the identification and characterization of intact protein complexes from native mass spectrometry data. © 2016 by John Wiley & Sons, Inc.

Keywords: protein complexes; multi‐proteoform complexes; top‐down proteomics; native mass spectrometry

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Characterizing a Multi‐Proteoform Complex with SEMPC
  • Alternate Protocol 1: Targeted Characterization of a Protein Complex not Present in the Corum Database
  • Guidelines for Understanding Results
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Belov, M.E., Damoc, E., Denisov, E., Compton, P.D., Horning, S., Makarov, A.A., and Kelleher, N.L. 2013. From protein complexes to subunit backbone fragments: A multi‐stage approach to native mass spectrometry. Anal. Chem. 85:11163‐11173. doi: 10.1021/ac4029328.
  Blackwell, A.E., Dodds, E.D., Bandarian, V., and Wysocki, V.H. 2011. Revealing the quaternary structure of a heterogeneous noncovalent protein complex through surface‐induced dissociation. Anal. Chem. 83:2862‐2865. doi: 10.1021/ac200452b.
  Havugimana, P.C., Hart, G.T., Nepusz, T., Yang, H., Turinsky, A.L., Li, Z., Wang, P.I., Boutz, D.R., Fong, V., Phanse, S., Babu, M., Craig, S.A., Hu, P., Wan, C., Vlasblom, J., Dar, V.‐u.‐N., Bezginov, A., Clark, G.W., Wu, G.C., Wodak, S.J., Tillier, E.R.M., Paccanaro, A., Marcotte, E.M., and Emili, A. 2012. A census of human soluble protein complexes. Cell 150:1068‐1081. doi: 10.1016/j.cell.2012.08.011.
  LeDuc, R.D., Fellers, R.T., Early, B.P., Greer, J.B., Thomas, P.M., and Kelleher, N.L. 2014. The C‐Score: A Bayesian framework to sharply improve proteoform scoring in high‐throughput top down proteomics. J. Proteome Res. 13:3231‐3240. doi: 10.1021/pr401277r.
  Liu, F., Rijkers, D.T.S., Post, H., and Heck, A.J.R. 2015. Proteome‐wide profiling of protein assemblies by cross‐linking mass spectrometry. Nat. Meth. 12:1179‐1184. doi: 10.1038/nmeth.3603.
  Meng, F., Cargile, B.J., Miller, L.M., Forbes, A.J., Johnson, J.R., and Kelleher, N.L. 2001. Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat. Biotech. 19:952‐957. doi: 10.1038/nbt1001‐952.
  Pesavento, J.J., Kim, Y.B., Taylor, G.K., and Kelleher, N.L. 2004. Shotgun annotation of histone modifications:  A new approach for streamlined characterization of proteins by top down mass spectrometry. J. Am. Chem. Soc. 126:3386‐3387. doi: 10.1021/ja039748i.
  Ruepp, A., Waegele, B., Lechner, M., Brauner, B., Dunger‐Kaltenbach, I., Fobo, G., Frishman, G., Montrone, C., and Mewes, H.‐W. 2010. CORUM: The comprehensive resource of mammalian protein complexes—2009. Nucleic Acids Res. 38:D497‐D501. doi: 10.1093/nar/gkp914.
  Skinner, O.S., Havugimana, P.C., Haverland, N.A., Fornelli, L., Early, B.P., Greer, J.B., Fellers, R.T., Durbin, K.R., Do Vale, L.H.F., Melani, R.D., Seckler, H.S., Nelp, M.T., Belov, M.E., Horning, S.R., Makarov, A.A., LeDuc, R.D., Bandarian, V., Compton, P.D., and Kelleher, N.L. 2016. An informatic framework for decoding protein complexes by top‐down mass spectrometry. Nat. Meth. 13:237‐240. doi: 10.1038/nmeth.3731. [published online ahead of print January 18, 2016].
  Smith, L.M. and Kelleher, N.L. 2013. Proteoform: A single term describing protein complexity. Nat. Methods 10:186‐187. doi: 10.1038/nmeth.2369.
  Tran, J.C., Zamdborg, L., Ahlf, D.R., Lee, J.E., Catherman, A.D., Durbin, K.R., Tipton, J.D., Vellaichamy, A., Kellie, J.F., Li, M., Wu, C., Sweet, S.M.M., Early, B.P., Siuti, N., LeDuc, R.D., Compton, P.D., Thomas, P.M., and Kelleher, N.L. 2011. Mapping intact protein isoforms in discovery mode using top‐down proteomics. Nature 480:254‐U141. doi: 10.1038/nature10575.
  Zamdborg, L., LeDuc, R.D., Glowacz, K.J., Kim, Y.B., Viswanathan, V., Spaulding, I.T., Early, B.P., Bluhm, E.J., Babai, S., and Kelleher, N.L. 2007. ProSight PTM 2.0: Improved protein identification and characterization for top down mass spectrometry. Nucleic Acids Res. 35:W701‐W706. doi: 10.1093/nar/gkm371.
PDF or HTML at Wiley Online Library