High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics
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. We report use of long (≥1 M) 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., 96.5 MPa or 14 K psi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5–5 μm 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 50 kDa. Furthermore, we chromatographed larger proteoforms (50–110 kDa) 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 <10 kDa 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 (200–400 Å) 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. Finally, 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.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1343947
- Alternate ID(s):
- OSTI ID: 1415303
- Report Number(s):
- PNNL-SA-107406; PII: S0021967317300225
- Journal Information:
- Journal of Chromatography, Vol. 1498, Issue C; ISSN 0021-9673
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Similar Records
Ultrahigh-Throughput Proteomics Using Fast RPLC Separations with ESI-MS/MS
Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments