High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics
•Column length was found as an important factor for top-down proteomic RPLC separation.•Long (≥1m) columns can provide peak capacities of >400 for resolving proteoforms.•Both porous and superficially porous particles were effective to separate proteins. Particles with 200–450Å pores enabled chrom...
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| Veröffentlicht in: | Journal of Chromatography Jg. 1498; H. C; S. 99 - 110 |
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| Hauptverfasser: | , , , , , , , , , |
| Format: | Journal Article |
| Sprache: | Englisch |
| Veröffentlicht: |
Netherlands
Elsevier B.V
19.05.2017
Elsevier |
| Schlagworte: | |
| ISSN: | 0021-9673, 1873-3778, 1873-3778 |
| Online-Zugang: | Volltext |
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| Zusammenfassung: | •Column length was found as an important factor for top-down proteomic RPLC separation.•Long (≥1m) columns can provide peak capacities of >400 for resolving proteoforms.•Both porous and superficially porous particles were effective to separate proteins. Particles with 200–450Å pores enabled chromatographing >100kDa proteoforms.•C1-C18-bonded phases had their own limits for eluting various sizes of proteoforms.
Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. Herein, we report use of long (≥1M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5MPa or 14Kpsi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5–5μm particles were used as packings and long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50kDa. Larger proteoforms (50–110kDa) were chromatographed on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200–400Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ∼900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC–MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Our initial data indicate that MS detection and fragmentation inefficiencies provided by current high-resolution mass spectrometers are key challenges for characterization of larger proteoforms. |
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| Bibliographie: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 AC05-76RL01830 PNNL-SA-107406 USDOE Office of Science (SC), Biological and Environmental Research (BER) |
| ISSN: | 0021-9673 1873-3778 1873-3778 |
| DOI: | 10.1016/j.chroma.2017.01.008 |