Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma

• Published in: Journal of proteome research Vol. 12; no. 12; pp. 5996 - 6003
• Main Authors: Lin, De, Alborn, William E, Slebos, Robbert J. C, Liebler, Daniel C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.12.2013
• Subjects: Amino Acid Sequence; Animals; Antibodies - chemistry; Antigens, CD - blood; Antigens, CD - genetics; Biomarkers, Tumor - blood; Biomarkers, Tumor - genetics; Carcinoma - blood; Carcinoma - diagnosis; Carcinoma - genetics; Cartilage Oligomeric Matrix Protein - blood; Cartilage Oligomeric Matrix Protein - genetics; Cattle; Colonic Neoplasms - blood; Colonic Neoplasms - diagnosis; Colonic Neoplasms - genetics; Endoglin; Enzyme-Linked Immunosorbent Assay - statistics & numerical data; GPI-Linked Proteins - blood; GPI-Linked Proteins - genetics; Hernia - blood; Hernia - diagnosis; Hernia - genetics; Humans; Immunoprecipitation - statistics & numerical data; Mass Spectrometry - methods; Matrix Metalloproteinase 9 - blood; Matrix Metalloproteinase 9 - genetics; Molecular Sequence Data; Receptors, Cell Surface - blood; Receptors, Cell Surface - genetics; Technical Note; Thrombospondins - blood; Thrombospondins - genetics; Tissue Inhibitor of Metalloproteinase-1 - blood; Tissue Inhibitor of Metalloproteinase-1 - genetics; MRM; plasma biomarkers; biomarker verification; colon cancer; immunoprecipitation
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr400877e

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.