Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping

Nature Protocols
Kunhong XiaoSheng Li

Abstract

Many cellular functions necessitate structural assemblies of two or more associated proteins. The structural characterization of protein complexes using standard methods, such as X-ray crystallography, is challenging. Herein, we describe an orthogonal approach using hydrogen-deuterium-exchange mass spectrometry (HDXMS), cross-linking mass spectrometry (CXMS), and disulfide trapping to map interactions within protein complexes. HDXMS measures changes in solvent accessibility and hydrogen bonding upon complex formation; a decrease in HDX rate could account for newly formed intermolecular or intramolecular interactions. To distinguish between inter- and intramolecular interactions, we use a CXMS method to determine the position of direct interface regions by trapping intermolecular residues in close proximity to various cross-linkers (e.g., disuccinimidyl adipate (DSA)) of different lengths and reactive groups. Both MS-based experiments are performed on high-resolution mass spectrometers (e.g., an Orbitrap Elite hybrid mass spectrometer). The physiological relevance of the interactions identified through HDXMS and CXMS is investigated by transiently co-expressing cysteine mutant pairs, one mutant on each protein at the discovered ...Continue Reading

References

Oct 15, 1992·Analytical Chemistry·G Thévenon-EmericD L Smith
Oct 18, 1990·Nature·M Karplus, G A Petsko
Mar 1, 1984·Nature·M AdrianA W McDowall
Sep 1, 1993·Proteins·Y BaiS W Englander
Apr 1, 1993·Protein Science : a Publication of the Protein Society·Z Zhang, D L Smith
Nov 15, 1996·Structure·M Sattler, S W Fesik
Dec 11, 1999·Nature·L H HartwellA W Murray
May 17, 2001·Proceedings of the National Academy of Sciences of the United States of America·P GhanouniB K Kobilka
Oct 31, 2001·Nature Structural Biology·K Wüthrich
May 30, 2003·Proceedings of the National Academy of Sciences of the United States of America·Joan J EnglanderVirgil L Woods
Oct 27, 2004·The Journal of Biological Chemistry·Kunhong XiaoRobert J Lefkowitz
Dec 8, 2004·Archives of Biochemistry and Biophysics·Laura S Busenlehner, Richard N Armstrong
Oct 7, 2005·Mass Spectrometry Reviews·Thomas E Wales, John R Engen
Oct 8, 2005·Journal of Structural and Functional Genomics·Huamin Zhang, William A Cramer
Jan 13, 2006·Methods in Enzymology·Claudia S Maier, Max L Deinzer
Apr 26, 2006·Molecular & Cellular Proteomics : MCP·Wilhelm HaasSteven P Gygi
Jun 27, 2006·Nature Chemical Biology·Xiaojie YaoBrian Kobilka
Aug 1, 2006·Journal of the American Society for Mass Spectrometry·S Walter Englander
Aug 10, 2006·Nature Methods·Michael J RustXiaowei Zhuang
Aug 12, 2006·Science·Eric BetzigHarald F Hess
Apr 5, 2007·Nature Protocols·Andrej ShevchenkoMatthias Mann
Jul 11, 2007·Proceedings of the National Academy of Sciences of the United States of America·Kunhong XiaoRobert J Lefkowitz
Oct 27, 2007·Protein Science : a Publication of the Protein Society·Lukasz SlabinskiAdam Godzik
Jan 4, 2008·Journal of the American Chemical Society·Kasper D RandThomas J D Jørgensen
Jan 16, 2008·Trends in Pharmacological Sciences·Brian Kobilka, Gebhard F X Schertler
May 20, 2008·Nature Methods·Roman SchmidtStefan W Hell
Jun 4, 2008·Physical Review Letters·D HannekeG Gabrielse
Oct 8, 2008·Molecular & Cellular Proteomics : MCP·Evgeniy V PetrotchenkoChristoph H Borchers
Nov 5, 2008·Journal of the American Chemical Society·Rinaldo W MontalvaoMichele Vendruscolo
Nov 19, 2008·Methods : a Companion to Methods in Enzymology·Christopher J McKeeMamuka Kvaratskhelia
Nov 19, 2008·Mass Spectrometry Reviews·Vanessa Leah Mendoza, Richard W Vachet
Apr 28, 2009·Physical Review Letters·Rolf ErniUlrich Dahmen

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Citations

Jan 31, 2020·Proteomics. Clinical Applications·Lei SongKunhong Xiao
Jan 30, 2021·The FEBS Journal·Aleš HnízdaTom L Blundell
Oct 29, 2020·Analytical Chemistry·John R EngenThomas E Wales
Dec 17, 2019·Journal of Medicinal Chemistry·Xinlian LiuChunyang Cao

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