Analyses of Individual Singly Charged Ions Using a High-Field Orbitrap Analyzer

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Biophysics

Background:

• Orbitrap-based charge detection mass spectrometry (CDMS) analyzes biomolecules at the individual-ion level.
• Longer transient recordings improve resolution and sensitivity in CDMS.
• Singly charged analytes (1-2 kDa) were previously undetectable due to low signal-to-noise ratios (S/N).

Purpose of the Study:

• To overcome the limitations of standard Orbitrap CDMS for singly charged analytes.
• To implement and evaluate CDMS on a high-field Orbitrap (HF-OT) mass analyzer for extended mass-to-charge (m/z) ranges.
• To establish new lower mass and charge limits for Orbitrap-based CDMS.

Main Methods:

• Utilized a high-field Orbitrap (HF-OT, Exploris 480) mass spectrometer coupled to an external data acquisition system.
• Adapted the mass spectrometer for robust detection across an extended m/z range (1 kDa to 500 kDa).
• Enabled transient recording up to 20 seconds to enhance signal detection.

Main Results:

• The HF-OT analyzer demonstrated superior charge and mass accuracy compared to a standard Orbitrap (S-OT) for various systems (insulin, BSA, monoclonal antibody).
• Achieved unprecedented mass resolution exceeding 3 million at m/z ~4250.
• Successfully detected singly charged peptides (angiotensin I, bradykinin) above the noise threshold (S/N = 2) for the first time, establishing new lower mass and charge limits.

Conclusions:

• The modified HF-OT analyzer significantly enhances resolution and S/N, enabling the detection of low-mass, low-charge biomolecules.
• This advancement expands the applicability of Orbitrap-based CDMS to a wider range of analytes, including peptides.
• The study sets a new benchmark for sensitivity and mass accuracy in individual-ion mass analysis.