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Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CHCl3, hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30,000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophosphatidylinositol, confirming membrane lipid extraction from the tissues.

Direct analysis of tissue by MALDI-MS allows the acquisition of its biomolecular profile while maintaining the integrity of the tissue, giving cellular localization, and avoiding tedious extraction and purification steps. However, direct tissue analysis generally leads to some extent to a lowered spectral quality due to variation in thickness, freezing tissue date, and nature of the tissue. We present here new technical developments for the direct tissue analysis of peptides with ionic liquid made of matrix mixtures (alpha-cyano-4-hydroxycinnamic acid (CHCA)/2-amino-4-methyl-5-nitropyridine and alpha-cyano-4-hydroxycinnamic acid/N,N-dimethylaniline (CHCA/DANI)). The properties of these direct tissue analysis matrixes, especially CHCA/aniline when compared to CHCA, 2,5-dihydroxybenzoic acid, and sinapinic acid, are as follows: (1) better spectral quality in terms of resolution, sensitivity, intensity, noise, number of compounds detected, and contaminant tolerance, (2) better crystallization on tissues, i.e., coverage capacity, homogeneity of crystallization, homogeneity of crystal sizes, and time of crystallization, (3) better analysis duration in term of vacuum stability, (4) better resistance to laser irradiation especially for high-frequency lasers, (5) better ionic yield in negative mode, and (6) enough fragmentation yield to use the PSD mode on sections to get structural information. Applied to MALDI imaging on a MALDI LIFT-TOF with a 50-Hz laser frequency, these ionic matrixes have allowed the realization of a new type of image in both polarities and reflector mode using the same tissue section. These results give a new outlook on peptide tissue profiling by MS, characterization of compounds from tissue slices, and MALDI-MS high-quality imaging.

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Internal energies and energy distributions were studied using the 'survival yield' method developed previously. In addition to conventional benzylpyridinium salts, protonated esters (fragmenting by rearrangement) and protonated leucine enkephalin were also used, extending the validity of the technique. Fragmentation processes were studied in the cone voltage region and modeled by the RRKM-based MassKinetics program. The results show that the shapes of the energy distributions are similar to thermal distributions. The mean internal energies are very similar for all compound classes studied, and show a linear increase with collision energy in the 10-50 eV region.

Recently, high-resolution mass spectrometry has been largely employed for compound identification, thanks to accurate mass measurements. As additional information, relative isotope abundance (RIA) is often needed to reduce the number of candidates prior to tandem MS(n). Here, we report on the evaluation of the LTQ-Orbitrap, in terms of accurate mass and RIA measurements for building further metabolomics spectral databases. Accurate mass measurements were achieved in the ppm range, using external calibration within 24 h, and remained at <5 ppm over a one-week period. The experimental relative abundances of (M+1) isotopic ions were evaluated in different data sets. First of all, 137 solutions of commercial compounds were analyzed by flow injection analysis in both the positive and negative ion modes. It was found that the ion abundance was the main factor impacting the accuracy of RIA measurements. It was possible to define some intensity thresholds above which errors were systematically <20% of their theoretical values. The same type of results were obtained with analyses from two biological media. Otherwise, no significant effect of ion transmission between the LTQ ion trap and the Orbitrap analyzer on RIA measurement errors was found, whereas the reliability of RIA measurements was dramatically improved by reducing the mass detection window. It was also observed that the signal integration method had a significant impact on RIA measurement errors, with the most-reliable results being obtained with peak height integrations. Finally, automatic integrations using the data preprocessing software XCMS and MZmine gave results similar to those obtained by manual integration, suggesting that it is relevant to use the RIA information in automatic elemental composition determination software from metabolomic peak tables.

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