Comprehensive identification of proteins from MALDI imaging.
Ontology highlight
ABSTRACT:
SUBMITTER: Maier SK
PROVIDER: S-EPMC3790299 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1,400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. This work presents a generic method and near complete list of MALDI IMS biomarkers that will become a valuable resource for the IMS community. Detailed description of the bioinformatics pipeline can be found in pipeline.txt. Briefly, different Mascot Distiller, Mascot and Scaffold versions have been used for the bottom-up and top-down data.
2013-07-03 | PXD000125 | Pride
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Project description:Autosomal recessive polycystic kidney disease is a severe, monogenetically inherited kidney and liver disease and PCK rats carrying the orthologous mutant gene serve as a model of human disease. We combined selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis of imaging data sets for identification of candidate lipid markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. Beside increased levels of serum-cholesterol these sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases suggesting that they be molecular markers of the disease. Genome-scale gene expression profiling of PCK and SD livers as control was performed to attempt elucidation of some of the underlying mechanisms leading to increases of cholesterol and taurine-conjugated bile acids in the PCK rat. Several pathways were found to be changed in cystic livers with up regulation or down regulation of important gene sets. Enhanced expression of steroid biosynthesis genes might result in the observed increased levels of cholesterol. In contrast, primary bile acid biosynthesis was found to be down regulated in diseased livers. These findings might be explained by compensatory mechanisms of liver metabolism to reduce toxic levels of accumulated bile acids. Snap-frozen liver tissue of 10 week old rats were subjected for RNA extraction and hybridization on Affymetrix microarrays to perform genome-scale gene expression profiling of n = 6 diseased PCK and n = 6 Sprague Dawley rat livers as control.
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Project description:Normalization is critically important for the proper interpretation of matrix-assisted laser desorption/ionization (MALDI) imaging datasets. The effects of the commonly used normalization techniques based on total ion count (TIC) or vector norm normalization are significant, and they are frequently beneficial. In certain cases, however, these normalization algorithms may produce misleading results and possibly lead to wrong conclusions, e.g. regarding to potential biomarker distributions. This is typical for tissues in which signals of prominent abundance are present in confined areas, such as insulin in the pancreas or β-amyloid peptides in the brain. In this work, we investigated whether normalization can be improved if dominant signals are excluded from the calculation. Because manual interaction with the data (e.g., defining the abundant signals) is not desired for routine analysis, we investigated two alternatives: normalization on the spectra noise level or on the median of signal intensities in the spectrum. Normalization on the median and the noise level was found to be significantly more robust against artifact generation compared to normalization on the TIC. Therefore, we propose to include these normalization methods in the standard "toolbox" of MALDI imaging for reliable results under conditions of automation.
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Project description:Autosomal recessive polycystic kidney disease is a severe, monogenetically inherited kidney and liver disease and PCK rats carrying the orthologous mutant gene serve as a model of human disease. We combined selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis of imaging data sets for identification of candidate lipid markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. Beside increased levels of serum-cholesterol these sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases suggesting that they be molecular markers of the disease. Genome-scale gene expression profiling of PCK and SD livers as control was performed to attempt elucidation of some of the underlying mechanisms leading to increases of cholesterol and taurine-conjugated bile acids in the PCK rat. Several pathways were found to be changed in cystic livers with up regulation or down regulation of important gene sets. Enhanced expression of steroid biosynthesis genes might result in the observed increased levels of cholesterol. In contrast, primary bile acid biosynthesis was found to be down regulated in diseased livers. These findings might be explained by compensatory mechanisms of liver metabolism to reduce toxic levels of accumulated bile acids.
2013-12-01 | GSE48357 | GEO
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Project description:Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is one of the most widely used methods for imaging the spatial distribution of unlabeled small molecules such as metabolites, lipids and drugs in tissues. Recent progress has enabled many improvements including the ability to achieve single cell spatial resolution, 3D-tissue image reconstruction, and the precise identification of different isomeric and isobaric molecules. However, MALDI-MSI of high molecular weight intact proteins in biospecimens has thus far been difficult to achieve. Conventional methods normally require in situ proteolysis and peptide mass fingerprinting, have low spatial resolution, and typically detect only the most highly abundant proteins in an untargeted manner. In addition, MSI-based multiomic and multimodal workflows are needed which can image both small molecules and intact proteins from the same tissue. Such a capability can provide a more comprehensive understanding of the vast complexity of biological systems at the organ, tissue, and cellular levels of both normal and pathological function. A recently introduced top-down spatial imaging approach known as MALDI HiPLEX-IHC (MALDI-IHC for short) provides a basis for achieving this high-information content imaging of tissues and even individual cells. Based on novel photocleavable mass-tags conjugated to antibody probes, high-plex, multimodal and multiomic MALDI-based workflows have been developed to image both small molecules and intact proteins on the same tissue sample. Dual-labeled antibody probes enable multimodal mass spectrometry and fluorescent imaging of targeted intact proteins. A similar approach using the same photocleavable mass-tags can be applied to lectin and other probes. We detail here several examples of MALDI-IHC workflows designed to enable high-plex, multiomic and multimodal imaging of tissues at a spatial resolution as low as 5 µm. This approach is compared to other existing high-plex methods such as imaging mass cytometry, MIBI-TOF, GeoMx and CODEX. Finally, future applications of MALDI-IHC are discussed.
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