title: Monitoring cytoplasmic protein complexes with blue native gel electrophoresis and stable isotope labelling with amino acids in cell culture: analysis of changes in the 20S proteasome.
abstract: Analysis of protein complexes is of increasing interest in the field of proteomics. A challenge is to develop methods for monitoring changes in the quantity and subunit composition of protein complexes on a proteome-wide scale. Here, we describe the combination of 1-D blue native polyacrylamide gel electrophoresis (BN-PAGE) with stable isotope labelling of amino acids in cell culture (SILAC) and tandem mass spectrometry (MS/MS). Cleared lysates from normal and perturbed samples, one incorporating heavy stable isotopes and the other light isotopes, are co-separated by blue native PAGE and then analysed and quantitated with MS/MS and appropriate software. This permits the analysis of cytoplasmic complexes. To demonstrate this technique, we explored how the 20S proteasome changes when the Pre9/α3 subunit, the only non-essential subunit of this complex, was deleted. Our results showed that ΔPre9/α3 cells can form the 20S proteasome complex, although with reduced efficiency. This involves an increase in expression of the α4 subunit. Our findings suggest this technique as an approach for the study of quantitative and qualitative differences in protein complexes, from cleared cell lysates.
authors.affiliation: Systems Biology Initiative, University of New South Wales, NSW, Australia.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Couttas
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Raftery
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Erce
authors.forename: Marc R
authors.initials: MR
authors.lastname: Wilkins
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D008970:Molecular Weight; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex