Novel Data Independent MALDI Imaging Ion Mobility Acquisition for the Visualization and Identification of Proteins Directly From a Single Tissue Section

We report a data independent MALDI imaging acquisition method where MS and MS/MS information are acquired within a single experiment, without any precursor ion selection. Post-acquisition, the precursors and the fragments are correlated on the basis of their common drift time, which is further refined utilizing the commonality of ion spatial distributions.

Initial experiments were carried out using thin sections of rat brain mounted on standard microscope slides. In order to digest the proteins directly from tissue, several coats of a trypsin solution were applied using a SunCollect nebulising spray device. After overnight incubation at 37°C, CHCA matrix was evenly applied. MS data were acquired using an ion mobility-enabled MALDI SYNAPT G2 MS. Within the same experiment, the mass spectrometer was set to apply alternate collision energies to the transfer cell between low energy (precursor ion data) and an elevated collision energy ramp, with the latter inducing peptide fragmentation. As fragmentation occurs after ion mobility separation, the precursors at low energy have the same drift time as their associated fragments from the elevated energy experiments. The experiment was designed such that adjacent pixels had low and elevated collision energy applied. Using novel HDI MALDI software, the elevated and low energy functions of the MALDI imaging data were independently processed with Apex 3D detection software. The low and high energy functions were independently visualised within the Analysis tab of the HDI software. Using the drift time alignment functionality, tryptic peptide precursors were associated with product ions that share similar drift times. For additional and supplementary refinement, another level of correlation was achieved on the basis of spatial distribution commonality.

Further work will be conducted and shown with the automatically HDI generated output (i.e. pkl file) for the automatic sequence annotation of multiple multiplex peptide spectra and identification of proteins from a single MALDI imaging experiment.