Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.
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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23536786/pdf/?tool=EBI |
| _version_ | 1818723829290106880 |
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| author | Jason D Russell Mark Scalf Adam J Book Daniel T Ladror Richard D Vierstra Lloyd M Smith Joshua J Coon |
| author_facet | Jason D Russell Mark Scalf Adam J Book Daniel T Ladror Richard D Vierstra Lloyd M Smith Joshua J Coon |
| author_sort | Jason D Russell |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-17T21:16:44Z |
| format | Article |
| id | doaj.art-e869f5a04c9f4a619d7c5c0ade520859 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-17T21:16:44Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-e869f5a04c9f4a619d7c5c0ade5208592022-12-21T21:32:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0183e5815710.1371/journal.pone.0058157Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.Jason D RussellMark ScalfAdam J BookDaniel T LadrorRichard D VierstraLloyd M SmithJoshua J CoonQuantification 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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23536786/pdf/?tool=EBI |
| spellingShingle | Jason D Russell Mark Scalf Adam J Book Daniel T Ladror Richard D Vierstra Lloyd M Smith Joshua J Coon Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry. PLoS ONE |
| title | Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry. |
| title_full | Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry. |
| title_fullStr | Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry. |
| title_full_unstemmed | Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry. |
| title_short | Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry. |
| title_sort | characterization and quantification of intact 26s proteasome proteins by real time measurement of intrinsic fluorescence prior to top down mass spectrometry |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23536786/pdf/?tool=EBI |
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