Abstract：
N-Terminal pro-B-type natriuretic peptide (NT-proBNP) is a pivotal biomarker for the diagnosis and prognosis of heart failure (HF). However, no SI-traceable certified reference material (CRM) or reference measurement procedure (RMP) is available for this biomarker, and so clinical testing results obtained in different laboratories cannot be traced to a higher-order standard, leading to incomparable measurements. Protein hydrolysis and protein cleavage isotope dilution mass spectrometry (AAA-IDMS and PepA-IDMS) were used to develop a CRM. Structurally related impurities were identified by high-resolution mass spectrometry. The quantitative AAA-IDMS results were corrected according to the amino acid compositions of the impurities. Using PepA-IDMS, two peptides from the proteolyzed product were confirmed as signature peptides. To obtain traceable and accurate results, the signature peptides were quantified using impurity-corrected AAA-IDMS. The candidate NT-proBNP solution was denatured and enzymatically digested using the Glu-C endoproteinase. The released signature peptides were measured using an isotopic dilution approach. The homogeneity and stability of the candidate CRM were characterized, and their uncertainties were combined with the value assignment process. The developed CRM can be considered a unique SI-traceable NT-proBNP reference material and is expected to be used as a primary calibrator for matrix NT-proBNP CRM development.
摘要：
N末端B型利钠肽原(NT-proBNP)是心力衰竭(HF)诊断和预后的重要生物标志物。然而,没有SI可追溯的认证参考材料（CRM）或参考测量程序（RMP）可用于此生物标志物，因此，在不同实验室获得的临床试验结果不能追溯到更高的标准，导致无与伦比的测量。蛋白质水解和蛋白质裂解同位素稀释质谱(AAA-IDMS和PepA-IDMS)用于开发CRM。通过高分辨率质谱鉴定结构相关的杂质。根据杂质的氨基酸组成校正定量AAA-IDMS结果。使用PepA-IDMS，来自蛋白水解产物的两种肽被证实为特征肽。为了获得可追溯和准确的结果，使用杂质校正的AAA-IDMS对特征肽进行定量.将候选NT-proBNP溶液变性并使用Glu-C内切蛋白酶酶消化。使用同位素稀释方法测量释放的特征肽。对候选CRM的均匀性和稳定性进行了表征，并将其不确定性与价值分配过程相结合。开发的CRM可以被认为是独特的SI可追溯的NT-proBNP参考材料，并有望用作矩阵NT-proBNPCRM开发的主要校准物。
