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A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections.

ABSTRACT: The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small sample sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities.

SUBMITTER: Acland M

PROVIDER: S-EPMC9326557 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections.

Acland Mitchell M Mittal Parul P Arentz Georgia G Whitehead Fergus F Hoffmann Peter P Klingler-Hoffmann Manuela M Oehler Martin K MK

Methods and protocols 20220710 4

The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass sp ...[more]

PMID: 35893583

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Project description:miRNA expression analysis was done on Uveal melanoma, metastatic and non - metastatic case in formalin fixed paraffin embedded sections, identified from case registry and confirmed by Chromosome insitu hybridization harboring monosomy 3 aberration in metastaic case and no aberration in non metastatic case. 19 miRNAs were found to be expressed only in class I tumors (choroidal melanoma) and not in class II (liver metastasis) and 11 miRNAs were found to be expressed only in class II and not in class I. The tumors were found to harbor oncomirs in both choroidal melanoma and metastasizing melanoma targeting tumor suppressor genes and metastatic suppressor genes. None of the differentially expressed miRNAs in either case were found to be located on the chromosomes which have been proved to carry chromosomal abnormalities. Rather it was found that genes targeted by the miRNAs were found to be present in chromosomal regions that are often found to be deleted 8p22, 13q and 17p. Organism used: Homo sapiens. * Slides: Human miRNA V2 8x15k Arrays AMADID: 19118. * Starting material: 18 slides with formalin fixed Paraffin embedded sections-20 μm. * RNA Samples used: 225-94-DUP, 225-94-E, 727-07-DUP, 727-07 -E. * Labeling kit: Agilent miRNA labeling reagent and Hybridization Kit. * Labeling Method: Ligation of one cyanine3-pCp molecule to the 3' end of RNA molecule with greater than 90% efficiency that generates fluorescent miRNA. * Total RNA and cRNA Purification Kit: Biorad MicroBio Spin 6 columns. * Hybridization Kit: Agilent miRNA Hybridization kit. Hybridization protocol See Agilent Technologies website for miRNA microarray hybridization and wash protocol: http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=50500 Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2505C) at 100% PMT with 10% for lower intensity (XDR Scanning).Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol Data processing Feature extracted data was analyzed using GeneSpring GX v 10.0.2 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended Percentile shift normalization (50th percentile) . Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. High expressed miRNA's were clustered using gene tree to identify significant gene expression patterns.

Dataset Information

A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections.

Publications

A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections.

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