Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid-chromatography-multiple- reaction-monitoring mass spectrometry.
Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid-chromatography-multiple- reaction-monitoring mass spectrometry.
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DOI：
10.1373/clinchem.2010.152264
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发表时间：
2010-12
影响因子：
9.3
通讯作者：
Hoofnagle AN
中科院分区：
文献类型：
--
作者：
Agger SA;Marney LC;Hoofnagle AN
如果liquid-chromatography–multiple-reaction–monitoring-MS能够用于大规模的生物标志物的临床前验证，将不再需要开发昂贵的免疫分析方法。此外，如果临床前研究中使用的分析方法与临床实验室中使用的分析方法相同，那么新的生物标记物将加速转化为临床使用。为了验证这一方法，我们开发了一种多重分析方法，用于定量测定人血浆中两种临床已知的生物标志物，载脂蛋白A-I和载脂蛋白B(apoA-I和apoB)。我们使用多肽图谱来鉴定候选多肽。人体样本用尿素或三氟乙醇变性，还原和烷化，然后用胰酶消化。我们比较了反相色谱分离多肽与正常流动和微流，并将内源性多肽峰面积归一化为内标肽。我们评估了不同的校准方法，并将最终的方法与散射比浊法进行了比较。我们开发了一种最终的方法，使用三氟乙醇变性，21h消化，正常流动色谱-电喷雾电离，并用单个正常人体血浆样本进行校准。对于两次注射的样品，两种蛋白的批内和批间变异系数分别为6%和12%，与免疫法(apoA-I和LC-MRM/MS=1.21×免疫分析+7.0；Sx|y=7.9)比较，差异均有统计学意义(n=47；Deming回归，LC-MRM/MS=1.17×免疫分析-36.6；Sx|y=10.3)。用LC-MRM/MS对人血浆/血清中的蛋白质进行多重定量是可能的，并与临床上有用的免疫分析方法进行了很好的比较。单点校准在大型临床研究中的潜在应用可以简化降低日常消化变异性的努力。
If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.
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影响因子：
9.3
作者：
Abbatiello SE;Mani DR;Keshishian H;Carr SA
通讯作者：
Carr SA
影响因子：
4.4
作者：
Anderson, NL;Anderson, NG;Pearson, TW
通讯作者：
Pearson, TW
影响因子：
9.3
作者：
Neubert, Hendrik;Gale, Jeremy;Muirhead, David
通讯作者：
Muirhead, David
影响因子：
7
作者：
Kuzyk, Michael A.;Smith, Derek;Borchers, Christoph H.
通讯作者：
Borchers, Christoph H.
影响因子：
3.4
作者：
Deutsch, EW;Eng, JK;Aebersold, R
通讯作者：
Aebersold, R