Comparison of protein immunoprecipitation-multiple reaction monitoring with ELISA for assay of biomarker candidates in plasma.
Comparison of protein immunoprecipitation-multiple reaction monitoring with ELISA for assay of biomarker candidates in plasma.
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DOI：
10.1021/pr400877e
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发表时间：
2013-12-06
影响因子：
4.4
通讯作者：
Liebler DC
中科院分区：
文献类型：
--
作者：
Lin D;Alborn WE;Slebos RJ;Liebler DC
血浆中蛋白质生物标志物的定量分析通常通过ELISA进行，但这种方法受到高质量抗体可用性的限制。另一种方法是蛋白质免疫沉淀结合多反应监测质谱法（IP-MRM）。我们比较了IP-MRM和ELISA用于分析结肠癌患者和非癌症对照血浆中的六种结肠癌生物标志物候选物（金属蛋白酶抑制剂1（TIMP 1）、软骨寡聚基质蛋白（COMP）、血小板反应蛋白-2（THBS 2）、内皮糖蛋白（ENG）、间皮素（MSLN）和基质金属蛋白酶-9（MMP 9））。用ELISA捕获抗体通过多重免疫沉淀从血浆中分析蛋白质，通过SDS-PAGE进一步纯化，消化并通过稳定同位素稀释MRM分析。IP-MRM提供了加标到由60 mg/mL牛血清白蛋白组成的“模拟血浆”基质中的靶蛋白在10和640 ng/mL之间的线性响应（r = 0.978-0.995）。IP-MRM测定的测量变异（检测限的变异系数）范围为2.3%至19%，与相同样品的ELISA的变异相似。除ENG外，所有靶蛋白的IP-MRM和ELISA测量值高度相关（r = 0.67-0.97）。因此，具有高质量捕获抗体的IP-MRM提供了用于生物流体中蛋白质定量的ELISA的有效替代方法。
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.
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影响因子：
9.3
作者：
Klee EW;Bondar OP;Goodmanson MK;Dyer RB;Erdogan S;Bergstralh EJ;Bergen HR 3rd;Sebo TJ;Klee GG
通讯作者：
Klee GG
影响因子：
7
作者：
Whiteaker, Jeffrey R.;Zhao, Lei;Paulovich, Amanda G.
通讯作者：
Paulovich, Amanda G.
影响因子：
9.9
作者：
Lange, Vinzenz;Picotti, Paola;Domon, Bruno;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi
影响因子：
7.4
作者：
Berna, Michael J.;Zhen, Yuejun;Ackermann, Bradley L.
通讯作者：
Ackermann, Bradley L.
影响因子：
4.4
作者：
Anderson, NL;Anderson, NG;Pearson, TW
通讯作者：
Pearson, TW