Exploring Charge-Detection Mass Spectrometry on Chromatographic Time Scales

[Image: see text] Charge-detection mass spectrometry (CDMS) enables direct measurement of the charge of an ion alongside its mass-to-charge ratio. CDMS offers unique capabilities for the analysis of samples where isotopic resolution or the separation of charge states cannot be achieved, i.e., heterogeneous macromolecules or highly complex mixtures. CDMS is usually performed using static nano-electrospray ionization-based direct infusion with acquisition times in the range of several tens of minutes to hours. Whether CDMS analysis is also attainable on shorter time scales, e.g., comparable to chromatographic peak widths, has not yet been extensively investigated. In this contribution, we probed the compatibility of CDMS with online liquid chromatography interfacing. Size exclusion chromatography was coupled to CDMS for separation and mass determination of a mixture of transferrin and β-galactosidase. Molecular masses obtained were compared to results from mass spectrometry based on ion ensembles. A relationship between the number of CDMS spectra acquired and the achievable mass accuracy was established. Both proteins were found to be confidently identified using CDMS spectra obtained from a single chromatographic run when peak widths in the range of 1.4–2.5 min, translating to 140–180 spectra per protein were achieved. After demonstration of the proof of concept, the approach was tested for the characterization of the highly complex glycoprotein α-1-acid glycoprotein and the Fc-fusion protein etanercept. With chromatographic peak widths of approximately 3 min, translating to ∼200 spectra, both proteins were successfully identified, demonstrating applicability for samples of high inherent molecular complexity.