Comparison of protein immunoprecipitation-multiple reaction monitoring with ELISA for assay of biomarker candidates in plasma.
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.
Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States.
De
D
Lin
William E
WE
Alborn
Robbert J C
RJ
Slebos
Daniel C
DC
Liebler
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D015703:Antigens, CD / Q000097:blood / Q000235:genetics; D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D002277:Carcinoma / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D064236:Cartilage Oligomeric Matrix Protein / Q000097:blood / Q000235:genetics; D002417:Cattle; D003110:Colonic Neoplasms / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D000071063:Endoglin; D004797:Enzyme-Linked Immunosorbent Assay / Q000706:statistics & numerical data*; D058851:GPI-Linked Proteins / Q000097:blood / Q000235:genetics; D006547:Hernia / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation / Q000706:statistics & numerical data*; D013058:Mass Spectrometry / Q000379:methods; D020780:Matrix Metalloproteinase 9 / Q000097:blood / Q000235:genetics; D000090204:Mesothelin; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000097:blood / Q000235:genetics; D019699:Thrombospondins / Q000097:blood / Q000235:genetics; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood / Q000235:genetics; D000099262:Thrombospondin 2
D000906:Antibodies; D015703:Antigens, CD; D014408:Biomarkers, Tumor; D064236:Cartilage Oligomeric Matrix Protein; D000071063:Endoglin; D058851:GPI-Linked Proteins; D020780:Matrix Metalloproteinase 9; D000090204:Mesothelin; D011956:Receptors, Cell Surface; D019699:Thrombospondins; D019715:Tissue Inhibitor of Metalloproteinase-1; D000099262:Thrombospondin 2; C064206:ENG protein, human; C000715527:MSLN protein, human; C560822:TIMP1 protein, human; C579270:MMP9 protein, human; C577553:TSP5 protein, human