Institute of Clinical Biochemistry, Innsbruck Medical University, Innsbruck , Austria.
Institute of Laboratory Medicine, Kantonsspital Aarau AG, Aarau, Switzerland.
Clin Chem. 2020 Sep 1;66(9):1200-1209. doi: 10.1093/clinchem/hvaa130.
The high molecular complexity of variably O-glycosylated and degraded pro B-type natriuretic peptide (proBNP) derived molecular forms challenges current immunoassays. Antibodies used show pronounced differences in cross-reactivities with these circulating fragments, which still need to be better characterized on a molecular level. To pave the way for advanced quantitative assays in the future, it is critical to fully understand these circulating forms.
Plasma samples were collected from 8 heart failure (HF) patients and 2 healthy controls. NT-proBNP and proBNP were purified by immunoprecipitation and analyzed by nano-flow liquid chromatography coupled to high-resolution mass spectrometry. Fragments formed during proteolysis in solution digestion were distinguished from naturally occurring peptides by using an 18O stable isotope labeling strategy.
We detected 16 previously unknown circulating fragments of proBNP peptides (9 of which are located in the N-terminal and 7 in the C-terminal region), revealing a more advanced state of degradation than previously known. Two of these fragments are indicative of either unidentified processing modes or a far-reaching C-terminal degradation (or a combination thereof) of the precursor proBNP.
Our results further restrict ideal target epitopes for immunoassay antibodies and expand the current thinking of diversity, degradation, and processing of proBNP, as well as the distribution of circulating forms.
具有可变 O-糖基化和降解的 B 型利钠肽原(proBNP)衍生分子形式的高分子复杂性给当前的免疫测定带来了挑战。所使用的抗体在与这些循环片段的交叉反应活性方面表现出明显的差异，这些差异仍需要在分子水平上更好地描述。为了为未来的先进定量测定铺平道路，充分了解这些循环形式至关重要。
从 8 例心力衰竭（HF）患者和 2 例健康对照中采集血浆样本。NT-proBNP 和 proBNP 通过免疫沉淀进行纯化，并通过纳流液相色谱与高分辨率质谱联用进行分析。在溶液消化过程中形成的片段通过使用 18O 稳定同位素标记策略与天然存在的肽区分开来。
我们检测到 proBNP 肽的 16 个以前未知的循环片段（其中 9 个位于 N 端，7 个位于 C 端区域），揭示了比以前已知的更先进的降解状态。其中两个片段表明存在未识别的加工模式或前体 proBNP 的广泛 C 端降解（或两者的组合）。
我们的研究结果进一步限制了免疫测定抗体的理想靶抗原表位，并扩展了 proBNP 的多样性、降解和加工以及循环形式的分布的现有思维。
