Title: High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics

Abstract

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 andmore » 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.« less

Authors:

Shen, Yufeng; Tolić, Nikola; Piehowski, Paul D.; Shukla, Anil K.; Kim, Sangtae; Zhao, Rui; Qu, Yi; Robinson, Errol; Smith, Richard D.; Paša-Tolić, Ljiljana

Publication Date:

Thu Jan 05 00:00:00 EST 2017

Research Org.:

Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1343947

Alternate Identifier(s):

OSTI ID: 1415303

Report Number(s):

PNNL-SA-107406
Journal ID: ISSN 0021-9673; PII: S0021967317300225

Grant/Contract Number:  

AC05-76RL01830

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Chromatography

Additional Journal Information:

Journal Volume: 1498; Journal Issue: C; Journal ID: ISSN 0021-9673

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; top-down proteomics; intact proteins; proteoforms; UPLC; mass spectrometry; columns and stationary phases