Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry

PLoS One. 2013;8(3):e58157. doi: 10.1371/journal.pone.0058157. Epub 2013 Mar 11.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / chemistry
  • Arabidopsis Proteins / analysis
  • Arabidopsis Proteins / chemistry
  • Carrier Proteins / analysis
  • Carrier Proteins / chemistry
  • Fluorescence
  • Mass Spectrometry* / methods
  • Proteasome Endopeptidase Complex / analysis
  • Proteasome Endopeptidase Complex / chemistry*
  • Proteins / analysis
  • Proteins / chemistry*
  • Reproducibility of Results
  • Tandem Mass Spectrometry


  • Arabidopsis Proteins
  • Carrier Proteins
  • Dss1 protein, Arabidopsis
  • Proteins
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease