Foulon North, Goonatilleke Elisha, MacCoss Michael J, Emrick Michelle A, Hoofnagle Andrew N
Department of Laboratory Medicine & Pathology, University of Washington, Seattle, WA, USA.
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
J Mass Spectrom Adv Clin Lab. 2022 Jun 10;25:19-26. doi: 10.1016/j.jmsacl.2022.06.003. eCollection 2022 Aug.
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.
胰岛素和C肽的测量为低血糖的临床评估提供了一种有价值的工具。在研究中，这些生物标志物一起用于更好地理解高胰岛素血症、肝脏胰岛素清除率和β细胞功能。液相色谱-串联质谱法（LC-MS/MS）是分析胰岛素和C肽的一种有吸引力的方法，因为该平台具有特异性，可避免免疫测定的某些局限性，并且可以进行多重分析。先前描述的用于同时定量胰岛素和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。这些方法在灵敏度和自身抗体干扰方面可能存在局限性。我们开发了一种新方法，该方法不需要抗体，而是使用蛋白水解消化产生易于离子化且能灵敏、特异且同时定量胰岛素和C肽的蛋白型肽段。
血清样本用乙腈沉淀。分析物通过固相萃取进行富集，然后用内肽酶Glu-C消化。使用靶向LC-MS/MS分析胰岛素和C肽的替代肽段。
日内不精密度低于20%CV，两种分析物（胰岛素=0.09 ng/mL，C肽=0.06 ng/mL）在定量下限以下均观察到线性。与市售胰岛素免疫测定法（贝克曼库尔特UniCel DxI 600 Access）比较，发现两种方法之间存在30%的偏差。
开发并评估了一种使用Glu-C消化同时分析胰岛素和C肽的新型LC-MS/MS方法。提供了详细的标准操作规程，以帮助其他实验室实施该方法。
