Deeper Protein Identification Using Field Asymmetric Ion Mobility Spectrometry in Top-Down Proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Field asymmetric ion mobility spectrometry (FAIMS) offers advanced gas-phase separation for proteomics.
• Top-down mass spectrometry is crucial for identifying and characterizing intact proteins and their proteoforms.
• Heterogeneous protein mixtures present challenges in comprehensive proteomic analysis.

Purpose of the Study:

• To evaluate the performance of cylindrical FAIMS for proteoform identification and characterization.
• To assess the impact of FAIMS coupled with chromatography on top-down mass spectrometry.
• To determine the utility of FAIMS in analyzing complex protein mixtures.

Main Methods:

• Utilized cylindrical FAIMS coupled with chromatographic separation.
• Performed top-down mass spectrometry on heterogeneous protein mixtures.
• Analyzed proteoform identification rates and signal-to-noise ratios with and without FAIMS.

Main Results:

• FAIMS combined with chromatography increased protein identifications by 62% and proteoform identifications by 8%.
• FAIMS improved proteoform identification accuracy and enabled detection of low-abundance proteins.
• Enhanced signal-to-noise ratios were observed for proteoforms with similar retention times.
• Optimal compensation voltage correlated with analyte molecular weight.

Conclusions:

• FAIMS significantly enhances the capabilities of top-down proteomics.
• The addition of FAIMS improves both discovery and targeted proteomic applications.
• FAIMS provides a valuable tool for detailed proteoform analysis in complex biological samples.