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Optimization and comparison of multiple MALDI matrix application methods for small molecule mass spectrometric imaging.

ABSTRACT: The matrix application technique is critical to the success of a matrix-assisted laser desorption/ionization (MALDI) experiment. This work presents a systematic study aiming to evaluate three different matrix application techniques for MALDI mass spectrometric imaging (MSI) of endogenous metabolites from legume plant, Medicago truncatula, root nodules. Airbrush, automatic sprayer, and sublimation matrix application methods were optimized individually for detection of metabolites in the positive ionization mode exploiting the two most widely used MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). Analytical reproducibility and analyte diffusion were examined and compared side-by-side for each method. When using DHB, the optimized method developed for the automatic matrix sprayer system resulted in approximately double the number of metabolites detected when compared to sublimation and airbrush. The automatic sprayer method also showed more reproducible results and less analyte diffusion than the airbrush method. Sublimation matrix deposition yielded high spatial resolution and reproducibility but fewer analytes in the higher m/z range (500-1000 m/z). When the samples were placed in a humidity chamber after sublimation, there was enhanced detection of higher mass metabolites but increased analyte diffusion in the lower mass range. When using CHCA, the optimized automatic sprayer method and humidified sublimation method resulted in double the number of metabolites detected compared to standard airbrush method.

SUBMITTER: Gemperline E

PROVIDER: S-EPMC4204912 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of lipids and proteins by using principal component analysis-linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study, we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis, and telomere stress induced senescence. In parallel, we also identified colocalization of hypoxic regions and various lipid species such as PC(16:0/18:0), PC(16:0/18:1), PC(16:0/18:2), PC(16:1/18:4), PC(18:0/18:1), and PC(18:1/18:1), among others. Our findings shed light on the biomolecular composition of hypoxic tumor regions, which may be responsible for a given tumor's resistance to radiation or chemotherapy.

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Optimization and comparison of multiple MALDI matrix application methods for small molecule mass spectrometric imaging.

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