Imaging of Similar Mass Neuropeptides in Neuronal Tissue by Enhanced Resolution MALDI MS with an Ion Trap OrbitrapTM Hybrid Instrument

Several mass spectrometry imaging (MSI) procedures are used to localize physiologically active peptides in neuronal tissue from American cockroach (Periplaneta americana ) neurosecretory organs. We report how to use this model system to assess, for the first time, the performance of the MALDI LTQ Orbitrap™ XL mass spectrometer to perform MSI of secretory neuropeptides. The method involves the following steps: (1) rapid dissecting of neurosecretory tissue (i.e., insect neurohemal organ) in isotonic sucrose solution; (2) mounting the tissue on a glass slide; (3) controlled spraying of the air-dried tissue with concentrated MALDI matrix solution; (4) loading specimen into the MALDI source of a MSⁿ system equipped with an Orbitrap™ analyzer; (5) setting-up MSI methods by determining tissue areas of interest, spatial resolution, molecular mass range, and molecular mass resolution; (6) acquiring mass spectra; (7) analyzing data using ImageQuest™ MSI software to generate (single or composite) images of the distribution of peptide(s) of interest; (8) confirming the identity of selected peptides by MS² and/or MS ⁿ sequencing directly from imaged tissue sample. The results illustrate that high mass accuracy and high mass resolving power of the Orbitrap analyzer are achievable in analyses directly from tissue, such as in MSI experiments. Moreover the mass spectrometric instrumentation evaluated allows for both peptide localization and peptide identification/sequencing directly from tissue.