title: Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.
abstract: Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.
authors.affiliation: Department of Chemistry, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Russell
authors.forename: Mark
authors.initials: M
authors.lastname: Scalf
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Book
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Ladror
authors.forename: Richard D
authors.initials: RD
authors.lastname: Vierstra
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Smith
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry; D029681:Arabidopsis Proteins / Q000032:analysis / Q000737:chemistry; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry; D005453:Fluorescence; D013058:Mass Spectrometry / Q000379:methods; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000737:chemistry*; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D029681:Arabidopsis Proteins; D002352:Carrier Proteins; C510575:Dss1 protein, Arabidopsis; D011506:Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease
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