Protein Analysis by Electrospray-Orbital Frequency Analyzer with Charge Detection Mass Spectrometry Algorithm.

Area of Science:

• Analytical Chemistry
• Biophysics
• Mass Spectrometry

Background:

• Electrostatic ion trap mass analyzers, such as the orbital frequency analyzer (OFA), are crucial for Fourier Transform Mass Spectrometry (FTMS).
• OFA offers ultrahigh mass resolution and high ion charge capacity, essential for analyzing large biological particles.
• Charge detection mass spectrometry (CDMS) is employed for analyzing multiply charged proteins and other large biomolecules.

Purpose of the Study:

• To develop a data processing algorithm for analyzing the image charge signal of nonharmonic waveforms in OFA-based CDMS.
• To enable the detection of collisions, determination of ion lifetimes, and evaluation of charge and mass values for ions.
• To eliminate chemical noise and facilitate the measurement of protein content at very low concentrations.

Main Methods:

• A novel data processing algorithm was created to interpret the nonharmonic image charge signals from ions within an OFA.
• The algorithm detects ion-molecule collisions to determine ion lifetimes.
• Ions with lifetimes above a set threshold are used to evaluate charge and mass, filtering out noise.

Main Results:

• The algorithm successfully filters chemical noise from small molecules and protein fragments, enabling protein concentration measurements down to tens of nanomolars.
• Standard deviation of charge measurement ranges from 1.1 to 1.8 e for ions with oscillation lifetimes between 1 and 0.4 s.
• Lower OFA voltage settings lead to longer ion survival times and narrower mass peak widths, improving spectral resolution.

Conclusions:

• The developed algorithm significantly enhances the capability of OFA-based CDMS for analyzing large, multiply charged biomolecules.
• The method allows for precise determination of ion charge and mass, improving the detection limits for proteins.
• While OFA supports multiplexed CDMS, coexisting ions with similar m/z can interfere with charge determination, requiring careful data interpretation.