Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.
Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.
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DOI：
10.1111/jth.15003
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发表时间：
2020-10
期刊：
影响因子：
--
通讯作者：
Johnstone AL
中科院分区：
文献类型：
--
作者：
Thålin C;Aguilera K;Hall NW;Marunde MR;Burg JM;Rosell A;Daleskog M;Månsson M;Hisada Y;Meiners MJ;Sun ZW;Whelihan MF;Cheek MA;Howard SA;Saxena-Beem S;Noubouossie DF;Key NS;Sheikh SZ;Keogh MC;Cowles MW;Lundström S;Mackman N;Wallén H;Johnstone AL
最近的数据提出了瓜氨酸化组蛋白H3（H3Cit）的诊断和预后能力，中性粒细胞胞外陷阱（NET）的标志物，在病理条件下，如癌症和血栓形成。然而，目前的研究受到缺乏标准化检测的阻碍。我们的目的是开发一种可靠地定量人血浆中核小体H3Cit的测定方法。我们评估了体外酶促修饰组蛋白H3作为校准标准品的常见做法和可用肽内瓜氨酸抗体的特异性。基于我们的研究结果，我们开发并验证了一种新的检测方法来定量人血浆中的核小体H3Cit。我们发现，酶促瓜氨酸化H3蛋白受到高度酶依赖性批次变异性以及血浆不稳定性的影响。我们还证明，当针对一组含有不同组蛋白H3瓜氨酸化的半合成核小体进行测试时，大多数市售抗肽内瓜氨酸抗体对其报告的靶点显示出较差的特异性。最后，我们提出了一种新的检测方法，利用高度特异性的单克隆抗体和半合成的核小体，含有瓜氨酸代替精氨酸在组蛋白H3，精氨酸残基2，8和17（H3R2，8，17Cit）作为校准标准。该测定的严格验证表明其准确可靠地定量人血浆中核小体H3Cit水平的能力，与健康个体相比，癌症患者中的核小体H3Cit水平明显升高。我们使用定义的核小体对照的新方法能够可靠地定量人血浆中的H3Cit。该测定将广泛适用于研究组蛋白瓜氨酸在疾病中的作用及其作为生物标志物的效用。
Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.
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DOI：
10.1126/science.aao4227
发表时间：
2018-09-28
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Albrengues J;Shields MA;Ng D;Park CG;Ambrico A;Poindexter ME;Upadhyay P;Uyeminami DL;Pommier A;Küttner V;Bružas E;Maiorino L;Bautista C;Carmona EM;Gimotty PA;Fearon DT;Chang K;Lyons SK;Pinkerton KE;Trotman LC;Goldberg MS;Yeh JT;Egeblad M
通讯作者：
Egeblad M
影响因子：
7.3
作者：
Berger-Achituv S;Brinkmann V;Abed UA;Kühn LI;Ben-Ezra J;Elhasid R;Zychlinsky A
通讯作者：
Zychlinsky A
影响因子：
--
作者：
Al-Haidari, Amr A;Algethami, Nader;Thorlacius, Henrik
通讯作者：
Thorlacius, Henrik
DOI：
10.1083/jcb.201212110
发表时间：
2013-07-08
期刊：
The Journal of cell biology
影响因子：
--
作者：
Ivanov A;Pawlikowski J;Manoharan I;van Tuyn J;Nelson DM;Rai TS;Shah PP;Hewitt G;Korolchuk VI;Passos JF;Wu H;Berger SL;Adams PD
通讯作者：
Adams PD
影响因子：
15.9
作者：
Cools-Lartigue, Jonathan;Spicer, Jonathan;Ferri, Lorenzo
通讯作者：
Ferri, Lorenzo