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

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Top-down proteomics, analyzing intact proteins, faces challenges in separation resolution compared to bottom-up proteomics.
• Existing separation methods for intact proteins limit the comprehensive analysis of proteoforms.
• High-resolution separation is crucial for advancing intact protein analysis and understanding proteome complexity.

Purpose of the Study:

• To develop and validate a high-resolution liquid chromatography method for intact protein separation.
• To investigate the impact of column characteristics on the resolution of proteoforms.
• To enhance the capabilities of top-down proteomics for global proteome analysis.

Main Methods:

• Utilized long (≥1M) reversed-phase liquid chromatography (RPLC) columns packed with short alkyl (C1-C4) bonded phases.
• Operated columns at a pressure limit of 96.5 MPa (14 Kpsi) to achieve maximal resolution.
• Systematically investigated particle types, including porous, nonporous, and superficially porous surfaces, and their pore sizes.

Main Results:

• Long columns with C1-C4 phases achieved high-resolution RPLC for proteoforms <50 kDa, yielding peak capacities >400.
• Short alkyl phases (C1-C4) were more efficient for global proteoform separation than long alkyl phases (C8, C18) for <10 kDa proteoforms.
• Enabled confident identification of approximately 900 proteoforms from low-microgram samples using single RPLC-MS/MS analysis.

Conclusions:

• Long RPLC columns with short alkyl phases are critical for achieving high-resolution separation in top-down proteomics.
• The achieved RPLC performance approaches that of bottom-up proteomics, facilitating accurate mass tag approaches.
• MS detection and fragmentation inefficiencies remain challenges for characterizing larger proteoforms (>50 kDa).