New Imaging Mass Spectrometry Technique Tested on Proteins

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Scientists from Vanderbilt University in Nashville, Tennessee recently tested a new means of imaging mass spectrometry (IMS) for isotopically resolved intact proteins, publishing their work in the journal Analytical Chemistry (1).

Untargeted biomolecular tissue imaging has been largely fueled by evolutions in imaging mass spectrometry (IMS). Typical IMS experiments involve sampling and ionizing analytes directly from tissue surfaces along a coordinate plane, detecting the analytes with a mass spectrometer, and visualizing it as ion heat maps according to ion abundances. With these practices, IMS has been used to map distributions of metabolites, lipids, peptides, proteins, and glycans, and is complementary to histological methods (stained and antibody-based imaging). However, while on-tissue digestion strategies expand the range of potentially detectable proteins, the risk of losing biologically relevant proteoform information (post-translational modifications, protein sequence truncations, point mutations, and more) increases. Alternatively, if proteoform information is preserved during intact protein analysis, the size of detectable proteins is typically limited.

The most used ionization method for intact protein IMS is matrix-assisted laser desorption/ionization (MALDI), which generates low-charge state ions at relatively high mass-to-charge (m/z) ratios. As such, intact protein MALDI IMS experiments are typically performed using time-of-flight (TOF) mass spectrometers owing to their theoretically unlimited m/z range and high sensitivity. However, TOF instruments usually have limited mass resolving power during tissue analysis, especially with detection in linear mode. This results in convoluted spectra where few accurate protein and proteoform intact masses can be determined.

According to the scientists, “The future of intact protein IMS relies on developing instrumentation that combines speed, high lateral spatial resolution, and high spectral resolution” (1). For this study, the scientists demonstrated rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflection time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Hybrid qTOF mass spectrometers possess the desired characteristics while also having the potential to combine the benefits of Fourier transform–mass spectrometers (FT–MS) and TOF platforms. Unlike FT–MS systems, where instrument resolving power decreases with increasing m/z, TOF and qTOF mass spectrometers have relatively constant resolving powers across the m/z range. Increased instrument scan times are unnecessary to achieve high mass resolution at high m/v.

Protein isotope distributions were resolved for both protein standard mixtures and proteins detect from a whole-body mouse pup tissue section. Rapid 10 µm lateral spatial resolution IMS was perform on a rat brain tissue section at a rate of approximately 10 pixels per second, all while maintaining isotopic spectral resolution. Finally, proof-of-concept MALDI–TIMS data was acquired from a protein mixture, all to demonstrate the system’s ability to differentiate charge states by ion mobility.

These findings show that when untargeted protein IMS is performed on a qTOF mass spectrometer, rapid and high lateral spatial resolution imaging can be performed without sacrificing spectral resolving power. Additionally, preliminary studies show the potential for TIMS to simplify analysis by separating proteins based on charge state.

“The reduced data acquisition time afforded by performing protein MALDI IMS on a qTOF mass spectrometer lowers the barrier to routine collection of high spatial and spectral resolution protein IMS data,” the scientists wrote (1). There is more work to be done, such as developing methods using a common MALDI qTOF platform, but this and more can provide a new starting point for the IMS community to advance the field of spatial proteomics.

References

(1) Klein, D. R.; Rivera, E. S.; Caprioli, R. M.; Spraggins, J. M. Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer. Anal. Chem. 2024, 96 (13), 5065–5070. DOI: 10.1021/acs.analchem.3c05252

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