Two-Dimensional Separation Using High-pH and Low-pH Reversed Phase Liquid Chromatography for Top-down Proteomics.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Top-down proteomics requires advanced separation techniques for complex intact protein analysis.
• One-dimensional reversed-phase liquid chromatography (1D-RPLC) has limitations in coverage for highly complex samples.
• Orthogonal separation methods are crucial for improving proteome coverage and detection dynamic range.

Purpose of the Study:

• To evaluate a "salt-free" high-pH RPLC as an orthogonal separation dimension to conventional low-pH RPLC for top-down mass spectrometry (MS).
• To assess the improvement in intact protein and proteoform identification using a 2D RPLC-RPLC-MS/MS approach.

Main Methods:

• Comparison of low-pH (pH=2) and high-pH (pH=10) RPLC separations to confirm orthogonality.
• Offline 2D RPLC-RPLC-MS/MS analysis of intact *E. coli* samples.
• Evaluation of intact protein and proteoform identification yields.

Main Results:

• The 2D RPLC-RPLC top-down MS approach identified 365 proteins and 886 proteoforms.
• This represents a substantial increase compared to the 1D RPLC-MS approach, which identified 163 proteins and 328 proteoforms.
• High-pH and low-pH RPLC demonstrated good orthogonality.

Conclusions:

• The 2D RPLC-RPLC top-down MS approach significantly enhances the depth of top-down proteomics.
• This method leverages the resolving power of RPLC and mass spectrometry-compatible buffers for efficient sample handling.
• The approach holds great potential for in-depth proteomic studies of complex intact protein samples.