Coupling online size exclusion chromatography with charge detection-mass spectrometry using Hadamard transform multiplexing

25 September 2024, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Charge detection mass spectrometry (CD-MS) is a powerful technique for the analysis of large, heterogeneous biomolecules. By directly measuring the charge states of individual ions, CD-MS can measure the masses from spectra where conventional deconvolution approaches fail due to the lack of isotopic resolution or distinguishable charge states. However, CD-MS is inherently slow because hundreds or thousands of spectra need to be collected to produce adequate ion statistics. The slower speed of CD-MS complicates efforts to couple it with online separation techniques, which limit the number of spectra that can be acquired during a chromatographic peak. Here, we present the application of Hadamard transform multiplexing to online size exclusion chromatography (SEC) coupled with Orbitrap CD-MS, with a goal of using SEC for separating complex mixtures prior to CD-MS analysis. We developed a microcontroller to deliver pulsed injections from a large sample loop onto a SEC for online CD-MS analysis. Data showed a series of peaks spaced according to the pseudo-random injection sequence, which were demultiplexed with a Hadamard transform algorithm. The demultiplexed data revealed improved CD-MS signals while preserving retention time information. This multiplexing approach provides a general solution to the inherent incompatibilities of online separations and CD-MS detection that will enable a range of applications.

Keywords

Charge detection-mass spectrometry
Hadamard transform
Native mass spectrometry
Size exclusion chromatography

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