Coupling Online Size Exclusion Chromatography with Charge Detection-Mass Spectrometry Using Hadamard Transform...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Charge detection mass spectrometry (CD-MS) is crucial for analyzing large, heterogeneous biomolecules.
• CD-MS measures ion masses directly, overcoming limitations of conventional methods with poor isotopic resolution or charge states.
• The slow speed of CD-MS hinders coupling with online separation techniques like size exclusion chromatography (SEC).

Purpose of the Study:

• To apply Hadamard transform multiplexing to online SEC coupled with Orbitrap CD-MS.
• To enable SEC separation of complex mixtures before CD-MS analysis.
• To overcome the speed limitations of CD-MS in online separation workflows.

Main Methods:

• Development of a microcontroller for pulsed sample injections onto a SEC system.
• Online coupling of SEC with Orbitrap CD-MS.
• Application of Hadamard transform algorithms for data demultiplexing.

Main Results:

• Observed a series of peaks spaced according to a pseudorandom injection sequence.
• Demultiplexed data demonstrated improved CD-MS signals.
• Retention time information was successfully preserved during multiplexed analysis.

Conclusions:

• Hadamard transform multiplexing offers a general solution for integrating online separations with CD-MS.
• This approach enhances CD-MS signal quality and preserves separation information.
• The developed method enables broader applications for analyzing complex biological samples.