High-throughput ion spectroscopy using Hadamard transform multiplexing and high-resolution ion mobility separations
The exceptional sensitivity of infrared (IR) ion spectroscopy to subtle differences in molecular structure offers a great promise for its analytical application in molecular identification problems in glycomics and metabolomics, for example [1,2], especially when combined with a rapid separation technique such as ion mobility spectrometry (IMS). The remaining challenge for the incorporation of spectroscopy into analytical workflows is to be able to acquire spectra in a high-throughput manner, i.e., for multiple species at the same time and at high speed. For this, we developed a novel approach using Hadamard transform multiplexing which allows measuring IR spectra of all species separated by ion mobility in a single laser scan.
In our approach, ions are generated by nano-electrospray ionization, separated by ion mobility, and then multiple combinations of ion packets with different mobility are sent to the ion trap for spectroscopy and mass analysis. The packets are selected according to an optimal pseudorandom sequence of length n. Once n pseudorandom sequences have been sent (multiplexing step), this process is repeated multiple times at each laser wavelength step. Then, data analysis is performed to de-multiplex the data at each wavelength step and obtain the IR spectra of all species with different ion mobility and mass-to-charge ratio.
To demonstrate the approach, we employed a combination of ultrahigh-resolution ion mobility IMS  and cryogenic ion spectroscopy to obtain highly-resolved vibrational spectra of more than 20 peptides originating from bovine serum albumin tryptic digest (0.5 μM). The spectroscopic analysis in the NH/OH stretch frequency region was completed in just 22 minutes, and with a 2-fold improvement in the signal-to-noise ratio compared to conventional signal averaging approach. Similar results were also obtained using IRMPD spectroscopy in the ion trap held at room temperature, although this approach generated much broader IR absorption lineshapes. Moreover, we have analyzed several mixtures of isomeric disaccharides, as well as pentasaccharide isomers found in human milk. The presented multiplexing approach is particularly suitable for the analysis of relatively complex mixtures with a broad range of mobilities and can be easily implemented in various IMS-MS setups combined with ion trapping.
 Ahmed Ben Faleh, Stephan Warnke, and Thomas R. Rizzo, Analytical Chemistry, 2019, 91, 7, 4876-4882.
 Jonathan Martens, Giel Berden et al., Scientific Reports, 2017, 7, 3363.
 Liulin Deng, Ian K. Webb et al., Analytical Chemistry, 2017, 89, 8, 4628-4634.