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

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. 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.

UniChromCD for Demultiplexing Time-resolved Charge Detection-Mass Spectrometry Data

“…The instrumentation used to generate the example SEC-MS data presented in this manuscript is described in detail in a companion paper, 29 so only a brief description is provided here. βgalactosidase and GroEL were purchased from Sigma or expressed and purified as described previously.…”

“…CD-MS data were acquired in Direct Mass Technology (DMT) mode on a Q Exactive HF UHMR Orbitrap mass spectrometer (Thermo Scientific Instruments) using a resolution setting of 240,000 at m/z 400 (512 ms transient length) and fixed ion injection times between 50 and 200 ms. Additional details can be found in the accompanying manuscript. 29 The IM instrument was an atmospheric pressure dual-gate drift tube constructed from printed circuit boards, according to the design published by Clowers et al 31 A drift voltage of 4 kV was applied, corresponding to an electric field strength of 200 V/cm. The ion gates were controlled by applying a voltage of 75 V (front) or 85 V (back) above the standard potential at their respective locations using a pair of FET pulsers built by GAA Custom Engineering (Kennewick, WA).…”

“…The sample was injected according to a 5-bit sequence, as described in a companion paper. 29 When raw CD-MS data is imported for the first time, the three plots shown in Figure 3A, B, and C are automatically generated. The raw chromatogram (Figure 3A) appears noisy because each individual scan contains only a few ions.…”