Quantification of cystatin-C in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry

Abstract

Cystatin C (CysC)-based estimated glomerular filtration rate is an excellent marker for early diagnosis of Chronic Kidney Disease (CKD). Particle-enhanced nephelometric immunoassay (PENIA) and particle-enhanced turbidimetric immunoassay (PETIA) are commonly used methods for CysC quantification in clinical testing. However, both of them lack of specificity which mass spectrometry offers. In this paper, an isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method was established for quantification of CysC in human serum. After CysC denaturation, reduction and alkylation of cysteine residues, trypsin was added for proteolytic digestion of CysC. Specific peptide ALDFAVGEYNK was selected as surrogates for intact CysC protein, then quantified CysC by stable isotope-labelled internal standard peptide A[¹³C6¹⁵N]LDFAVGEYNK based on calibration curve method. The calibration curves were y=0.1565x-1.6715 (R²=0.988) and y=1.8785x-2.2497 (R²=0.991) for y⁹ and y⁶, respectively. The linear range was 0.1-10mg/L and 0.5-15mg/L for y⁹ and y⁶, respectively. The limit of quantification (LOQ) was 0.052mg/L. At different concentrations of CysC, the recoveries were in the range of 80.5%-93.7%, the intraday precisions were in the range of 0.3%-2.2%, and the inter-day precisions were in the range of 0.2%-2.8%. The results show that ID-LC-MS/MS and PETIA methods have higher consistency (y=0.8021x+0.6611, p=0.14), and the mean difference of the two methods was -0.29mg/L, and 95% of the individual difference values were in the range of -0.91mg/L-0.33mg/L.

Keywords: Calibration curve; Cystatin C; ID-LC–MS/MS; Proteolytic digestion.