High-efficiency nano- and micro-HPLC--high-resolution Orbitrap-MS platform for top-down proteomics.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Orbitrap mass analyzers offer high resolution, mass accuracy, and sensitivity.
• Ion-pair reversed-phase high-performance liquid chromatography (RP HPLC) is a common technique for separating biomolecules.

Purpose of the Study:

• To evaluate the potential of interfacing Orbitrap mass spectrometry (MS) with ion-pair RP HPLC for intact protein analysis.
• To assess the performance of monolithic columns compared to silica-based stationary phases for protein recovery and carryover.

Main Methods:

• Utilized Orbitrap-MS coupled with ion-pair RP HPLC using acetonitrile gradients and monolithic columns (1.0 and 0.10 mm id).
• Investigated protein detection limits, mass range, chromatographic peak widths, and isotopic resolution.
• Employed a micro-HPLC-Orbitrap-MS setup for monoclonal antibody characterization and nano-HPLC-Orbitrap-MS for subproteome analysis.

Main Results:

• Achieved peak capacities of 120-130 within 20-25 min separation times.
• Demonstrated superior protein recovery and reduced carryover with monolithic columns compared to silica-based phases.
• Detected intact proteins from 5.7-150 kDa with low femtomol limit of detection (LOD).
• Observed isotopic resolution for proteins up to 42 kDa.
• Successfully characterized a 150 kDa monoclonal antibody and analyzed a complex wheat grain subproteome.

Conclusions:

• The Orbitrap-MS and ion-pair RP HPLC interface is highly effective for intact protein analysis, offering high resolution and sensitivity.
• Monolithic columns provide significant advantages in protein recovery and reduced carryover for this application.
• The developed method is applicable to the analysis of complex protein mixtures, including large proteins and subproteomes.