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

Orbitrap-based charge detection mass spectrometry (CDMS) enables the mass analysis of biomolecules at the individual-ion level. Recording longer transients boosts resolution and sensitivity for CDMS measurements. However, singly charged analytes (∼1-2 kDa) remained elusive to Orbitrap-based CDMS due to their low signal-to-noise ratios (S/N). To overcome this limitation, we implemented CDMS on a high-field Orbitrap mass analyzer (HF-OT, Exploris 480 mass spectrometer) coupled to an external data acquisition system. The HF-OT analyzer should theoretically improve the resolution by 1.7-fold and the S/N by 1.4-fold compared to a standard Orbitrap analyzer (S-OT, UHMR mass spectrometer). We first adapted an Exploris 480 mass spectrometer to allow robust detection across an extended m/z range (∼1 kDa to 500 kDa) and enable transient recording up to 20 s. Experimentally, the HF-OT outperformed the S-OT in charge and mass accuracy for a variety of systems, such as insulin, BSA, and a monoclonal antibody, and allowed us to achieve an unprecedented mass resolution far above 3 million even at m/z ∼ 4250. On the lower side of the m/z range, the modified HF-OT allowed the recording of individual molecules carrying just a single charge. For the first time, the peptides angiotensin I and bradykinin could be unambiguously detected and mass analyzed above the noise threshold (S/N = 2), albeit only at extended transient times, creating a new lower mass and charge limit for Orbitrap-based CDMS.