Department of Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong, 226001, China.
Department of Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong, 226001, China.
Anal Biochem. 2019 Dec 15;587:113451. doi: 10.1016/j.ab.2019.113451. Epub 2019 Sep 25.
Cystatin C is considered as an alternative to the evaluation of glomerular filtration rate. In this study, we highlighted an LC-MS/MS approach for the absolute quantitation of serum cystatin C based on label-free internal standards. A tryptic peptide (ALDFAVGEYNK) was selected as the surrogate whilst analogue (ALDFAVGEYQK) served as an internal standard. After denaturation, reduction, alkylation, digestion and concentration, the target peptides were separated on an LC column and monitored under MRM. The calibration range was from 0.25 mg/L to 15 mg/L with LLOQ of 0.05 mg/L and LOD of 0.03 mg/L, respectively. The certified reference material (ERM-DA471) was determined at 5.12 mg/L with bias of 6.57%. The recovery was between 89.68% and 92.43%. The RSD of intra- and inter-assay imprecision were both <10%. Good stability was also observed. The assay also demonstrated that the quantification of native cystatin C in human serum could be achieved using label-free internal standards. The assay was robust, cheap and sensitive.
半胱氨酸蛋白酶抑制剂 C 被认为是评估肾小球滤过率的一种替代方法。在本研究中，我们强调了一种基于无标记内标物的 LC-MS/MS 方法，用于血清胱抑素 C 的绝对定量。选择胰酶肽 (ALDFAVGEYNK) 作为替代物，而类似物 (ALDFAVGEYQK) 则作为内标物。经过变性、还原、烷基化、消化和浓缩后，目标肽在 LC 柱上分离，并在 MRM 下监测。校准范围为 0.25mg/L 至 15mg/L，LLOQ 为 0.05mg/L，LOD 为 0.03mg/L。用 ERM-DA471 标准物质进行验证，结果为 5.12mg/L，偏差为 6.57%。回收率在 89.68%至 92.43%之间。日内和日间精密度的 RSD 均<10%。稳定性也很好。该方法还证明，可以使用无标记内标物对人血清中的天然胱抑素 C 进行定量。该测定方法具有稳健、廉价和灵敏的特点。
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