title: Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex.
abstract: Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science , Rehovot 7610001, Israel.
authors.forename: Gili
authors.initials: G
authors.lastname: Ben-Nissan
authors.affiliation: Thermo Fisher Scientific , 28199 Bremen, Germany.
authors.forename: Mikhail E
authors.initials: ME
authors.lastname: Belov
authors.affiliation: The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science , Rehovot 7610001, Israel.
authors.forename: David
authors.initials: D
authors.lastname: Morgenstern
authors.affiliation: The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science , Rehovot 7610001, Israel.
authors.forename: Yishai
authors.initials: Y
authors.lastname: Levin
authors.affiliation: Israel Structural Proteomics Center, Weizmann Institute of Science , Rehovot 7610001, Israel.
authors.forename: Orly
authors.initials: O
authors.lastname: Dym
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science , Rehovot 7610001, Israel.
authors.forename: Galina
authors.initials: G
authors.lastname: Arkind
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science , Rehovot 7610001, Israel.
authors.forename: Carni
authors.initials: C
authors.lastname: Lipson
authors.affiliation: Thermo Fisher Scientific , 28199 Bremen, Germany.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Makarov
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science , Rehovot 7610001, Israel.
authors.forename: Michal
authors.identifier: 0000-0003-3933-0595
authors.initials: M
authors.lastname: Sharon
mesh_terms: D006597:Fructose-Bisphosphatase / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D010766:Phosphorylation; D021122:Protein Subunits / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013696:Temperature
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C111172:FBP1 protein, S cerevisiae; D006597:Fructose-Bisphosphatase