Ion-Ion Charge Reduction Addresses Multiple Challenges Common to Denaturing Intact Mass Analysis

Area of Science:

• Proteomics
• Analytical Chemistry
• Mass Spectrometry

Background:

• Intact protein mass analysis is crucial for complete characterization.
• Protein overcharging and sample heterogeneity complicate conventional mass spectrometry (MS) analysis.
• Current methods struggle with unstructured proteins and complex mixtures.

Purpose of the Study:

• To introduce and validate a novel method, full scan proton transfer charge reduction (fsPTCR), for improved intact protein analysis.
• To overcome limitations of conventional MS1 analysis, including overcharging and heterogeneity.
• To demonstrate analytical advantages in sensitivity, proteoform identification, and quantitative accuracy.

Main Methods:

• Coupling rapid denaturing size exclusion chromatography with fsPTCR-Orbitrap MS.
• Utilizing time-resolved deconvolution for data analysis.
• Optimizing collisional gas pressure for protein retention/removal.

Main Results:

• fsPTCR significantly reduced overcharging and increased signal-to-noise (11-fold) for a flexible protein.
• Analysis of a glycoprotein revealed twice as many proteoforms compared to MS1.
• Increased sensitivity and quantitative accuracy were observed for a protein in a complex mixture.
• Collisional gas pressure was identified as a key parameter for spectral control.

Conclusions:

• fsPTCR offers substantial analytical advantages over conventional MS1 for intact protein characterization.
• The method effectively addresses challenges posed by protein overcharging and sample heterogeneity.
• fsPTCR provides a powerful strategy for enhanced proteomic analysis of diverse protein samples.