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Mass spectrometry based biomarker discovery, verification, and validation--quality assurance and control of protein biomarker assays.

ABSTRACT: In its early years, mass spectrometry (MS)-based proteomics focused on the cataloging of proteins found in different species or different tissues. By 2005, proteomics was being used for protein quantitation, typically based on "proteotypic" peptides which act as surrogates for the parent proteins. Biomarker discovery is usually done by non-targeted "shotgun" proteomics, using relative quantitation methods to determine protein expression changes that correlate with disease (output given as "up-or-down regulation" or "fold-increases"). MS-based techniques can also perform "absolute" quantitation which is required for clinical applications (output given as protein concentrations). Here we describe the differences between these methods, factors that affect the precision and accuracy of the results, and some examples of recent studies using MS-based proteomics to verify cancer-related biomarkers.

SUBMITTER: Parker CE

PROVIDER: S-EPMC5528535 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Mass spectrometry based biomarker discovery, verification, and validation--quality assurance and control of protein biomarker assays.

Parker Carol E CE Borchers Christoph H CH

Molecular oncology 20140320 4

In its early years, mass spectrometry (MS)-based proteomics focused on the cataloging of proteins found in different species or different tissues. By 2005, proteomics was being used for protein quantitation, typically based on "proteotypic" peptides which act as surrogates for the parent proteins. Biomarker discovery is usually done by non-targeted "shotgun" proteomics, using relative quantitation methods to determine protein expression changes that correlate with disease (output given as "up-or ...[more]

PMID: 24713096

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Project description:Targeted glycoproteomics represents an attractive approach for conducting peripheral blood based cancer biomarker discovery due to the well-known altered pattern of protein glycosylation in cancer and the reduced complexity of the resultant glycoproteome. Here we report its application to a set of pooled nonsmall cell lung cancer (NSCLC) case sera (9 adenocarcinoma and 6 squamous cell carcinoma pools from 54 patients) and matched controls pools, including 8 clinical control pools with computed tomography detected nodules but being nonmalignant as determined by biopsy from 54 patients, and 8 matched healthy control pools from 106 cancer-free subjects. The goal of the study is to discover biomarkers that may enable improved early detection and diagnosis of lung cancer. Immunoaffinity subtraction was used to first deplete the topmost abundant serum proteins; the remaining serum proteins were then subjected to hydrazide chemistry based glycoprotein capture and enrichment. Hydrazide resin in situ trypsin digestion was used to release nonglycosylated peptides. Formerly N-linked glycosylated peptides were released by peptide-N-glycosidase F (PNGase F) treatment and were subsequently analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A MATLAB based in-house tool was developed to facilitate retention time alignment across different LC-MS/MS runs, determination of precursor ion m/z values and elution profiles, and the integration of mass chromatograms based on determined parameters for identified peptides. A total of 38 glycopeptides from 22 different proteins were significantly differentially abundant across the case/control pools (P < 0.01, Student's t test) and their abundances led to a near complete separation of case and control pools based on hierarchical clustering. The differential abundances of three of these candidate proteins were verified by commercially available ELISAs applied in the pools. Strong positive correlations between glycopeptide mass chromatograms and ELISA-measured protein abundance was observed for all of the selected glycoproteins.

Dataset Information

Mass spectrometry based biomarker discovery, verification, and validation--quality assurance and control of protein biomarker assays.

Publications

Mass spectrometry based biomarker discovery, verification, and validation--quality assurance and control of protein biomarker assays.

Similar Datasets

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