Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies.
Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies.
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DOI：
10.1016/j.jmsacl.2022.06.003
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发表时间：
2022-08
影响因子：
2.2
通讯作者：
Hoofnagle, Andrew N.
中科院分区：
文献类型：
--
作者：
Foulon, North;Goonatilleke, Elisha;MacCoss, Michael J.;Emrick, Michelle A.;Hoofnagle, Andrew N.
无抗体同时定量检测胰岛素和C-肽。用Glu-C进行蛋白质分解，可以进行灵敏和精确的测量。使用经过认证的标准物质进行校准可提供可追溯性。与商业化的免疫分析相比，偏倚相对较大。胰岛素和C-肽的测定为临床评估低血糖提供了有价值的工具。在研究中，这些生物标记物一起用于更好地了解高胰岛素血症、肝脏胰岛素清除和β细胞功能。液-质联用(LC-MS/MS)是分析胰岛素和C-肽的一种很有吸引力的方法，因为其平台具有专一性，可以避免免疫分析的某些限制，并且可以多重使用。先前描述的同时定量胰岛素和C-肽的LC-MS/MS方法测量完整的分析物，并且大多数依赖于免疫亲和富集。这些方法可能分别在敏感性和自身抗体的干扰方面受到限制。我们开发了一种新的方法，不需要抗体，并使用蛋白水解酶产生容易电离的蛋白质型肽，使胰岛素和C-肽的敏感、特异和同时定量成为可能。血清样品用乙腈沉淀。样品用固相萃取富集，然后用内切酶Glu-C消化。用有针对性的LC-MS/MS分析胰岛素和C-肽的替代多肽，日内不精密度小于20%，两种分析物的线性关系都达到了下限(胰岛素和C-肽的含量分别为0.09和0.06 ng/mL)。与市售胰岛素免疫测定法(Beckman Coulter Unicel DXi 600 Access)相比，两种方法之间存在30%的偏差。建立了用Glu-C消化同时分析胰岛素和C-肽的LC-MS/MS方法，并对其进行了评价。提供了详细的标准操作程序，以帮助促进在其他实验室的实施。
Simultaneous quantification of insulin and C-peptide without antibodies. Proteolysis with Glu-C permits sensitive and precise measurements. Calibration with certified reference material provides traceability. Relatively large bias when compared with a commercially available immunoassay. The measurement of insulin and C-peptide provides a valuable tool for the clinical evaluation of hypoglycemia. In research, these biomarkers are used together to better understand hyperinsulinemia, hepatic insulin clearance, and beta cell function. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an attractive approach for the analysis of insulin and C-peptide because the platform is specific, can avoid certain limitations of immunoassays, and can be multiplexed. Previously described LC-MS/MS methods for the simultaneous quantification of insulin and C-peptide measure the intact analytes and most have relied on immunoaffinity enrichment. These approaches can be limited in terms of sensitivity and interference from auto-antibodies, respectively. We have developed a novel method that does not require antibodies and uses proteolytic digestion to yield readily ionizable proteotypic peptides that enables the sensitive, specific, and simultaneous quantitation of insulin and C-peptide. Serum samples were precipitated with acetonitrile. Analytes were enriched using solid phase extraction and then digested with endoproteinase Glu-C. Surrogate peptides for insulin and C-peptide were analyzed using targeted LC-MS/MS. Inter-day imprecision was below 20 %CV and linearity was observed down to the lower limit of quantitation for both analytes (insulin = 0.09 ng/mL, C-peptide = 0.06 ng/mL). Comparison to a commercially available insulin immunoassay (Beckman Coulter UniCel DxI 600 Access) revealed a 30% bias between methods. A novel LC-MS/MS method for the simultaneous analysis of insulin and C-peptide using Glu-C digestion was developed and evaluated. A detailed standard operating procedure is provided to help facilitate implementation in other laboratories.
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