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MALDI Imaging for Proteomic Painting of Heterogeneous Tissue Structures
摘要： Purpose: To present matrix-assisted laser desorption/ionization (MALDI) imaging as a powerful method to highlight various tissue compartments. Experimental design: Formalin-fixed paraffin-embedded (FFPE) tissue of a uterine cervix, a pancreas, a duodenum, a teratoma and a breast cancer tissue microarray (TMA) were analyzed by MALDI imaging and by immunohistochemistry (IHC). Peptide images were visualized and analyzed using FlexImaging and SCiLS Lab software. Different histological compartments were compared by hierarchical cluster analysis. Results: MALDI imaging highlights tissue compartments comparable to IHC. In cervical tissue, normal epithelium could be discerned from intraepithelial neoplasia. In pancreatic and duodenal tissues, m/z signals from lymph follicles, vessels, duodenal mucosa, normal pancreas and smooth muscle structures could be visualized. In teratoma, specific m/z signals to discriminate squamous epithelium, sebaceous glands, and soft tissue were detected. Additionally, tumor tissue could be discerned from the surrounding stroma in small tissue cores of TMAs. Proteomic data acquisition of complex tissue compartments in FFPE tissue required less than one hour with recent mass spectrometers. Conclusion and clinical relevance: The simultaneous characterization of morphological and proteomic features in the same tissue section adds proteomic information for histopathological diagnostics, which relies at present on conventional Hematoxylin and Eosin staining, histochemical, IHC and molecular methods.
关键词： FFPE,immunohistochemistry,Tumor heterogeneity,MALDI imaging
更新于2025-09-10 09:29:36
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Ultraviolet Photodissociation of ESI- and MALDI-Generated Protein Ions on a Q-Exactive Mass Spectrometer
摘要： The identification of molecular ions produced by MALDI or ESI strongly relies on their fragmentation to structurally informative fragments. The widely diffused fragmentation techniques for ESI multiply charged ions are either incompatible (ECD and ETD) or show lower efficiency (CID, HCD), with the predominantly singly charged peptide and protein ions formed by MALDI. In-source decay has been successfully adopted to sequence MALDI-generated ions, but it is not compatible with mass-spectrometry imaging. Excellent UVPD performances, in terms of number of fragment ions and sequence coverage, has been demonstrated for electrospray ionization for multiple proteomics applications. UVPD showed a much lower charge-state dependence, and so protein ions produced by MALDI may exhibit equal propensity to fragment. Here we report UVPD implementation on an Orbitrap Q-Exactive Plus mass spectrometer equipped with an ESI/EP-MALDI. UVPD of MALDI-generated ions was benchmarked against MALDI-ISD, MALDI-HCD, and ESI-UVPD. MALDI-UVPD outperformed MALDI-HCD and ISD, efficiently sequencing small proteins ions. Moreover, the singly charged nature of MALDI-UVPD avoids the bioinformatics challenges associated with highly congested ESI-UVPD mass spectra. Our results demonstrate the ability of UVPD to further improve tandem mass spectrometry capabilities for MALDI-generated protein ions. Data are available via ProteomeXchange with identifier PXD011526.
关键词： Q-Exactive,MALDI,MS/MS,ultraviolet photodissociation
更新于2025-09-10 09:29:36
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Pre-Processing MALDI/TOF Mass Spectra by Using Geena 2
摘要： Geena 2 is a tool for filtering, averaging, and aligning MALDI/TOF mass spectra, designed to assist scientists in the analysis of high volumes of data and support them for comparative studies. Three web interfaces are available with different levels of complexity. In this manuscript, we explain how to use Geena 2 with these three interfaces to perform analyses of one’s own data. Two support protocols showing how to check the example input file and how to create an input file with own data are also presented.
关键词： spectra alignment,mass spectrometry,pre-processing,MALDI/TOF
更新于2025-09-09 09:28:46
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Atmospheric Pressure MALDI-IMS Using p-Nitroaniline as the Matrix at High Spatial Resolution in the Positive and Negative Ion Modes
摘要： Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) can provide information on the spatial distribution and amount of numerous molecules on a single tissue section. A single sample that can be used in both positive and negative ion analyses is needed, since this can reduce losses of rare clinical samples. Although p-nitroaniline (NIT) has been shown to be applicable for use as a matrix in MALDI-IMS in the positive and negative ion modes, it is volatile and instable under vacuum conditions. In this study, we applied NIT as a volatile matrix for atmospheric pressure (AP) MALDI-IMS analysis, using a Mass Microscope, because NIT is considered to remain stable on the sample under AP conditions. Our objective was to optimize NIT usage by evaluating peak intensities acquired in the positive and negative ion modes. We evaluated signal intensities under 3 different conditions; dilution at different volumes, at different concentrations, and in two types of solvents. We also measured the matrix weight deposited on samples and determined the optimum condition with respect to matrix weight and signal intensity. The optimum condition was determined as follows; a solvent volume of 2 mL, an NIT concentration of 30 mg/mL, and 100% methanol as the solvent. Mass spectra obtained using these conditions provided comparable signals with 2,5-dihydroxybenzoic acid and 9-aminoacridine. Using tandem mass spectrometry, the most intense signals in both ion modes were identified as phosphatidylcholine and phosphatidylinositol. Moreover, the lipid distribution in the mouse cerebellum could be visualized at a high spatial resolution of 5 μm on a single section, in both the positive and negative ion modes. This study shows that optimization of NIT is determined by 3 parameters (solvent volume, NIT concentration, type of solvent). Using the optimum condition, it was possible to analyze the same tissue section by AP MALDI-IMS in both the positive and negative ion modes, at a sufficiently high sensitivity for tandem mass spectrometry and imaging at a spatial resolution of 5 μm.
关键词： AP MALDI,Positive and negative ion modes,p-Nitroaniline,Phospholipids,High spatial resolution,IMS
更新于2025-09-09 09:28:46
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Targeted Feature Extraction in MALDI Mass Spectrometry Imaging to Discriminate Proteomic Profiles of Breast and Ovarian Cancer
摘要： Purpose: To develop a mass spectrometry imaging (MSI) based workflow for extracting m/z values related to putative protein biomarkers and using these for reliable tumor classification. Experimental design: Given a list of putative breast and ovarian cancer biomarker proteins, we extracted a set of related m/z values from heterogeneous MSI datasets derived from formalin-fixed paraffin-embedded tissue material. Based on these features, a linear discriminant analysis classification model was trained to discriminate the two tumor types. Results: We show that the discriminative power of classification models based on the extracted features is increased compared to the automatic training approach, especially when classifiers are applied to spectral data acquired under different conditions (instrument, preparation, laboratory). Conclusions and clinical relevance: We obtained robust classification models not confounded by technical variation between MSI measurements. This supports the assumption that the classification of the respective tumor types is based on biological rather than technical differences, and that the selected features are related to the proteomic profiles of the tumor types under consideration.
关键词： feature extraction,tumor typing,MALDI MSI,tissue classification,biomarker proteins
更新于2025-09-09 09:28:46
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NOVEL MATRIX SUBLIMATION DEVICE FOR MALDI MASS SPECTROMETRY IMAGING
摘要： Sublimation is a widely used method for matrix deposition in imaging mass spectrometry experiments. Still, most of the time standard glass sublimators are used for this purpose, which do not enable optimal matrix deposition reproducibility, compromising inter-experiment comparison of the results. Here we present an in-house designed stainless steel sublimator in which the parameters that have the strongest influence over matrix deposition reproducibility can be easily monitored. Using sections of human colon biopsies we demonstrate the capabilities of this new prototype.
关键词： mass spectrometry imaging,matrix sublimation,stainless steel sublimator,reproducibility,MALDI
更新于2025-09-09 09:28:46
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SENSING IMMOBILIZED MOLECULES OF STREPTAVIDIN ON A SILICON SURFACE BY MALDI-TOF MASS SPECTROMETRY AND FLUORESCENCE MICROSCOPY
摘要： A hydrogen-terminated Si (111) surface was modified to form an aminoterminated monolayer for immobilization of streptavidin. Cleavage of an N-(ω-undecylenyl)-phthalimide covered surface using hidrazine yields an amino group-modified surface, which serves as a substrate for the attachment of biotin and subsequently streptavidin. We used surface analytical techniques to characterize the surface and to control the course of functionalization before the immobilization of streptavidin. To confirm the presence of the streptavidin Texas red on the surface two powerful techniques available in a standard biochemical laboratory are used, Fluorescence Microscopy and MALDI-TOF that allow us to detect and determine the immobilized streptavidin. This work provides an avenue for the development of devices in which the exquisite binding specificity of biomolecular recognition is directly coupled to a biosensor. In addition, we have demonstrated that MALDI-TOF and fluorescence microscopy are useful techniques for the characterization of silicon functionalized surfaces.
关键词： streptavidin,fluorescence microscopy,MALDI-TOF,biosensor,silicon surface
更新于2025-09-04 15:30:14