Ozcan Filiz, Akbas Halide, Kırac Ebru, Suleymanlar Gultekin, Aslan Mutay, Yucel Gultekin
Department of Biochemistry, Faculty of Medicine, Akdeniz University, Antalya, Turkey.
Division of Nephrology, Department of Internal Medicine, Faculty of Medicine, Akdeniz University, Antalya, Turkey.
Rapid Commun Mass Spectrom. 2016 Mar 15;30(5):603-10. doi: 10.1002/rcm.7474.
Urinary liver fatty acid binding protein (L-FABP) has been evaluated as a promising early biomarker of renal ischemia in human kidney transplant patients. The use of L-FABP in clinical practice requires that this biomarker be associated with an analytical method that combines specificity, accuracy and robustness. This study aimed to evaluate an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography coupled with tandem mass spectrometry to measure urinary L-FABP levels in renal transplant recipients.
Purified recombinant human L-FABP tryptic standard was analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS/MS) and liquid chromatography (LC)/MS/MS to select for peptides that provided specificity and adequate response in developing an MRM method for urinary L-FABP quantification. Human urine samples collected from kidney transplant recipients were isolated, concentrated, precipitated and trypsin digested before mass spectrometric analysis of L-FABP. L-FABP levels were also measured in urine samples by enzyme immunoassay.
The tryptic peptide ion MH(+) of (50) FTITAGSK(57) (m/z 824) provided an adequate signal and was used for quantification of L-FABP under conditions employed for LC/MS/MS analysis. MALDI-TOF-MS/MS spectra obtained by collision-induced dissociation of the parent MH(+) ion (50) FTITAGSK(57) resulted in a y3 product ion that was used for quantitative analysis by the MRM method. Urinary L-FABP content measured by both ELISA and LC/MS/MS after transplantation was significantly higher compared to before transplantation levels. The Spearman correlation coefficient between the two methods was statistically significant. Intra-day and inter-day coefficients of variation provided good repeatability and reproducibility for validation of LC/MS/MS analysis.
LC/MS/MS quantification of L-FABP may provide a new reference method to determine changes in this potential biomarker in human kidney transplant patients.
尿肝脂肪酸结合蛋白（L-FABP）已被评估为人类肾移植患者肾缺血的一种有前景的早期生物标志物。在临床实践中使用L-FABP需要这种生物标志物与一种结合了特异性、准确性和稳健性的分析方法相关联。本研究旨在评估一种优化的多反应监测（MRM）方法，该方法使用超快速液相色谱与串联质谱联用，以测量肾移植受者尿液中的L-FABP水平。
通过基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱（MS/MS）和液相色谱（LC）/MS/MS分析纯化的重组人L-FABP胰蛋白酶标准品，以选择在开发用于尿液L-FABP定量的MRM方法时提供特异性和足够响应的肽段。从肾移植受者收集的人类尿液样本在进行L-FABP的质谱分析之前，先进行分离、浓缩、沉淀和胰蛋白酶消化。还通过酶免疫测定法测量尿液样本中的L-FABP水平。
（50）FTITAGSK（57）的胰蛋白酶肽离子MH(+)（m/z 824）提供了足够的信号，并用于在LC/MS/MS分析所用条件下对L-FABP进行定量。通过对母离子MH(+)（50）FTITAGSK（57）进行碰撞诱导解离获得的MALDI-TOF-MS/MS光谱产生了一个y3产物离子，该离子用于通过MRM方法进行定量分析。移植后通过ELISA和LC/MS/MS测量的尿L-FABP含量均显著高于移植前水平。两种方法之间的Spearman相关系数具有统计学意义。日内和日间变异系数为LC/MS/MS分析的验证提供了良好的重复性和再现性。
LC/MS/MS对L-FABP的定量分析可能为确定人类肾移植患者中这种潜在生物标志物的变化提供一种新的参考方法。
