Abstract
Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978-0.995) between 10 and 640 ng/mL for the target proteins spiked into a "mock plasma" matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67-0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.
Publication types
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Comparative Study
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Sequence
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Animals
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Antibodies / chemistry
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Antigens, CD / blood
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Antigens, CD / genetics
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Biomarkers, Tumor / blood*
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Biomarkers, Tumor / genetics
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Carcinoma / blood*
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Carcinoma / diagnosis
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Carcinoma / genetics
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Cartilage Oligomeric Matrix Protein / blood
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Cartilage Oligomeric Matrix Protein / genetics
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Cattle
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Colonic Neoplasms / blood*
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Colonic Neoplasms / diagnosis
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Colonic Neoplasms / genetics
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Endoglin
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Enzyme-Linked Immunosorbent Assay / statistics & numerical data*
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GPI-Linked Proteins / blood
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GPI-Linked Proteins / genetics
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Hernia / blood
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Hernia / diagnosis
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Hernia / genetics
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Humans
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Immunoprecipitation / statistics & numerical data*
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Mass Spectrometry / methods
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Matrix Metalloproteinase 9 / blood
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Matrix Metalloproteinase 9 / genetics
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Mesothelin
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Molecular Sequence Data
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Receptors, Cell Surface / blood
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Receptors, Cell Surface / genetics
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Thrombospondins / blood
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Thrombospondins / genetics
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Tissue Inhibitor of Metalloproteinase-1 / blood
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Tissue Inhibitor of Metalloproteinase-1 / genetics
Substances
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Antibodies
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Antigens, CD
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Biomarkers, Tumor
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COMP protein, human
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Cartilage Oligomeric Matrix Protein
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ENG protein, human
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Endoglin
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GPI-Linked Proteins
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MSLN protein, human
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Receptors, Cell Surface
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TIMP1 protein, human
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Thrombospondins
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Tissue Inhibitor of Metalloproteinase-1
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thrombospondin 2
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MMP9 protein, human
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Matrix Metalloproteinase 9
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Mesothelin