Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Intact protein analysis using liquid chromatography-mass spectrometry (LC-MS) is advancing due to improved mass spectrometer capabilities.
• Simultaneous detection of post-translational modifications (PTMs) is crucial for monitoring proteoform status in clinical and production settings.
• Identifying PTMs on large proteins or those with multiple modification sites presents significant challenges.

Purpose of the Study:

• To investigate the impact of various instrumental parameters on spectral quality for intact protein LC-MS.
• To identify key parameters for optimizing the detection of low-abundance post-translational modifications (PTMs).
• To establish a methodological framework for robust proteoform identification.

Main Methods:

• Analysis of low-abundance proteoforms of C-reactive protein, vitamin D-binding protein, transferrin, and NISTmAb using an Orbitrap Elite mass spectrometer.
• Systematic investigation of instrument parameters: source temperatures, in-source CID, microscan settings, resolution, and automatic gain control.
• Evaluation of signal-to-noise ratio as a metric for spectral quality and PTM identification.

Main Results:

• Spectral quality was significantly influenced by instrument parameters, with signal-to-noise ratio proving effective for identifying low-abundance PTMs.
• Optimization of source temperature and CID voltage was protein-specific.
• Key instrumental parameters were identified for enhancing PTM detection via LC-MS.

Conclusions:

• Optimized LC-MS parameters are essential for improved detection of diverse PTMs on intact proteins.
• A robust methodological framework was established for reliable proteoform identification.
• This work provides a foundation for the ultimate quantification of proteoforms.