Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Proteomics research faces challenges due to the complexity of the proteome, requiring protein fractionation before mass spectrometry (MS) analysis.
• Separating intact proteins for top-down proteomics remains difficult despite advancements.
• Conventional hydrophobic interaction chromatography (HIC) offers high-resolution protein separation but uses MS-incompatible salts.

Purpose of the Study:

• To identify a mass spectrometry (MS)-compatible salt for hydrophobic interaction chromatography (HIC).
• To develop a novel two-dimensional (2D) chromatographic method for high-resolution intact protein separation.
• To demonstrate the utility of HIC coupled with reverse phase chromatography (RPC) for top-down proteomics.

Main Methods:

• Identified ammonium tartrate as an MS-compatible salt for HIC, showing comparable performance to ammonium sulfate.
• Demonstrated that ammonium tartrate provides selectivity orthogonal to reverse phase chromatography (RPC).
• Coupled HIC and RPC to create a 2D chromatographic method for intact protein separation.

Main Results:

• Ammonium tartrate was validated as an MS-compatible HIC mobile phase salt.
• The HIC-RPC 2D method achieved effective high-resolution separation of intact proteins from standard mixtures and cell lysates.
• Successful identification of separated intact proteins using high-resolution top-down MS.

Conclusions:

• Hydrophobic interaction chromatography (HIC) is a promising high-resolution separation technique for top-down proteomics.
• The novel 2D HIC-RPC method enables effective intact protein separation and identification.
• This approach significantly advances the capabilities of top-down proteomics analysis.