Considerations for quantification of lipids in nerve tissue using matrix-assisted laser desorption/ionization mass spectrometric imaging.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is a technique that provides the ability to identify and characterize endogenous and exogenous compounds spatially within tissue with relatively little sample preparation. While it is a proven methodology for qualitative analysis, little has been reported for its utility in quantitative measurements. In the current work, inherent challenges in MALDI quantification are addressed. Signal response is monitored over successive analyses of a single tissue section to minimize error due to variability in the laser, matrix application, and sample inhomogeneity. Methods for the application of an internal standard to tissue sections are evaluated and used to quantify endogenous lipids in nerve tissue. A precision of 5% or less standard error was achieved, illustrating that MALDI imaging offers a reliable means of in situ quantification for microgram-sized samples and requires minimal sample preparation.

Imaging of similar mass neuropeptides in neuronal tissue by enhanced resolution MALDI MS with an ion trap - Orbitrap 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 MS(n) 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 MS(2) 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.

Mass spectral analysis of neuropeptide expression and distribution in the nervous system of the lobster Homarus americanus.

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. A total of 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.