A hybrid linear ion trap/Orbitrap mass spectrometer was used to perform MS/MS experiments and high resolution mass analysis of lipids desorbed from nerve tissue. A dramatic improvement in mass spectral resolution and a decrease in background are observed in the spectra collected from the Orbitrap mass analyzer, which allow generation of more accurate mass spectrometric images of the distribution of lipids within nerve tissue. Employment of both mass analyzers provides a rapid and reliable means of compound identification based on MS/MS fragmentation and HRMS accurate mass.

Nanospray desorption electrospray ionization (nano-DESI) combined with tandem mass spectrometry (MS/MS), high-resolution mass analysis of the fragment ions (m/Δ=17,500 at m/z 200), and rapid spectral acquisition enabled simultaneous imaging and identification of a large number of metabolites and lipids from 92 selected m/z windows (± 1 Da) with a spatial resolution of better than 150 μm. Mouse uterine sections of implantation sites on day 6 of pregnancy were analyzed in the ambient environment without any sample pre-treatment. MS/MS imaging was performed by scanning the sample under the nano-DESI probe at 10 μm/s while acquiring higher-energy collision-induced dissociation (HCD) spectra for a targeted inclusion list of 92 m/z values at a rate of ~6.3 spectra/s. Molecular ions and their corresponding fragments, separated using high-resolution mass analysis, were assigned based on accurate mass measurement. Using this approach, we were able to identify and image both abundant and low-abundance isobaric and isomeric species within each m/z window. MS/MS analysis enabled efficient separation and identification of isomeric and isobaric phospholipids that are difficult to separate in a full-scan mode. Furthermore, we identified several metabolites associated with early pregnancy and obtained the first 2D images of these molecules.

The lobster Homarus americanus has long served as an important animal model for electrophysiological and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biological functions. Using nanoLC ESI Q-TOF MS/MS and three types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/extract analysis and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resolution and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide analysis in terms of peptidome coverage, sensitivity, mass spectral resolution and capability for de novo sequencing.