In this work, we demonstrate 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-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (~10 pixels/s) 10 μm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.
Figure S1. MALDI mass spectrum of red phosphorus; Figure S2. MALDI mass spectra of the protein standard as varying TIMS Funnel In pressures; Figure S3. MALDI mass spectrum averaged across the entire mouse pup tissue section; Figure S4. Mouse pup ion images; Table S1. List of red phosphorus ions used for instrument calibration; Table S2. Table of protein standards, including amino acid sequences; Equation S1. Equation for theoretical spectral acquisition time on a 15T FT ICR; Equation S2. Equation for theoretical total aqusution time for a 170,000 pixel MALDI IMS image on a 15T FT-ICR.