Exploring Charge-Detection Mass Spectrometry on Chromatographic Time Scales

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Charge-detection mass spectrometry (CDMS) directly measures ion charge and mass-to-charge ratio.
• CDMS is valuable for complex samples like heterogeneous macromolecules and mixtures where other methods fail.
• Current CDMS methods typically require long acquisition times (tens of minutes to hours).

Purpose of the Study:

• To investigate the feasibility of CDMS analysis on shorter timescales compatible with liquid chromatography.
• To assess the performance of online liquid chromatography-coupled CDMS for complex biological samples.

Main Methods:

• Coupling size exclusion chromatography (SEC) with CDMS for online separation and mass analysis.
• Analyzing mixtures of transferrin and β-galactosidase, followed by complex glycoprotein and Fc-fusion protein characterization.
• Establishing a correlation between the number of CDMS spectra and mass accuracy.

Main Results:

• Successful identification of transferrin and β-galactosidase using CDMS spectra from single chromatographic runs (140-180 spectra per protein).
• Demonstrated applicability to complex samples: α-1-acid glycoprotein and etanercept were identified with chromatograph peak widths of ~3 min (~200 spectra).
• Established a relationship between CDMS spectra count and achievable mass accuracy.

Conclusions:

• CDMS is compatible with online liquid chromatography, enabling rapid analysis of complex mixtures.
• This hyphenated technique significantly reduces analysis time while maintaining confident identification of proteins.
• The approach is suitable for characterizing highly complex glycoproteins and fusion proteins, expanding CDMS applications.