title: Exposing the subunit diversity within protein complexes: a mass spectrometry approach.
abstract: Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Shelly
authors.initials: S
authors.lastname: Rozen
authors.forename: Alessandra
authors.initials: A
authors.lastname: Tieri
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ridner
authors.forename: Ann-Kathrin
authors.initials: AK
authors.lastname: Stark
authors.forename: Tilo
authors.initials: T
authors.lastname: Schmaler
authors.forename: Gili
authors.initials: G
authors.lastname: Ben-Nissan
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Dubiel
authors.forename: Michal
authors.initials: M
authors.lastname: Sharon
mesh_terms: D000075686:COP9 Signalosome Complex; D005591:Chemical Fractionation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry*; D011489:Protein Denaturation; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods
chemical_list: D046912:Multiprotein Complexes; D021122:Protein Subunits; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex