Invention:
This technology uses a multiplexing approach to online size exclusion chromatography (SEC) coupled with Orbitrap CD-MS. A microcontroller is used to deliver pulsed injections from a large sample loop onto a SEC for online CD-MS analysis. The method produces a series of peaks spaced according to the pseudo-random injection sequence, which are demultiplexed with a Hadamard transform algorithm. This improves CD-MS signals while preserving the 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.
Background:
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 produce accurate mass measurements from spectra where conventional deconvolution approaches fail due to the lack of isotopic resolution or distinguishable charge states. CD-MS has found applications in the characterization of very large (up to tens of mega-Daltons (MDa)) biomolecules such as protein complexes, intact virus capsids, vaccines, and gene therapies. Growing needs in biotechnology and the introduction of commercial instruments has led to increased interest in analyzing large, heterogeneous biomolecules. CD-MS is limited in that only a small number of ions can be measured at a time, which necessitates the acquisition of many hundreds or thousands of spectra to provide enough detection events for accurate mass measurements with acceptable signal to noise ratios. These long acquisitions generally prevent the coupling of CD-MS with online (i.e., real-time) separation techniques, such as size exclusion chromatography (SEC), which typically only provide enough time for 50-200 scans during the elution of a chromatographic peak. Progress has been made on this front by refining CD-MS instrumentation to enable the measurement of multiple ions simultaneously and improve charge state assignments with deconvolution algorithms. However, a recent effort to evaluate the feasibility of online SEC-CD-MS produced deconvolved mass spectra that, while sufficient to measure accurate protein masses, had lower signal-to-noise ratios and contained significantly more erroneous peaks than spectra produced from infusion experiments. Accordingly, there remains a need to develop methods, and apparatuses containing a device, for improved acquisition of analytical data for very large, heterogeneous biomolecules and/or microorganisms.
Applications:
- Biomolecule design and characterization
- Bio-based product development for therapeutics or diagnostics
- QC, forensics
Advantages:
- Improved signal-to-noise while preserving retention time information
- Improvement to CD-MS methods
