Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure
Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure
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DOI：
10.1073/pnas.0508782102
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发表时间：
2005-11-29
影响因子：
11.1
通讯作者：
Muddiman, DC
中科院分区：
文献类型：
--
作者：
Hawkridge, AM;Heublein, DM;Muddiman, DC
脑c端（b型）利钠肽(BNP)-32是一种广泛应用于心衰（HF）诊断、预后和治疗的临床生物标志物。32-aa颗粒主要在心房和心室心肌中合成，构成未成熟BNP（前BNP）的成熟生物活性形式。越来越多的证据表明，BNP以不同的结构形式循环，影响HF的诊断和体内活性。在此，我们开发并使用了一种免疫亲和纯化方法，从纽约心脏协会IV级患者血浆中分离出内源性BNP-32，随后通过纳米液相色谱（LC）电喷雾电离傅立叶变换离子回旋共振（FT-ICR）质谱法进行分析。我们将稳定同位素标记的BNP-32引入测定血浆中，以实现内源性IBNP-32水平的量化。与世界范围内用于从血浆中定量BNP-32的化学非特异性护理点测试（POCTs）和RIAs不同，FT-ICR-MS（前所未有的质量测量精度）加上LC（保留时间）提供了非凡的分子特异性，并且当与使用内部标准相结合时能够自信地识别和定量BNP-32。这项工作的意义在于，尽管POCTs测定的纽约心脏协会IV类患者血液中BNP-32的循环水平极高（> 290 fmol/ml），纳米lc电喷雾电离- ft - icr - ms数据并未显示任何内源性BNP-32。这些结果为晚期HF患者缺乏循环BNP-32提供了分子特异性证据，并提示存在改变形式的BNP对POCT值有贡献。
C-terminal brain (B-type) natriuretic pepticle (BNP)-32 is a widely used clinical biomarker for the diagnosis, prognosis, and treatment of heart failure (HF). The 32-aa pepticle is synthesized primarily in the atrial and ventricular myocardium and constitutes the mature biologically active form of immature BNP (pro-BNP). There has been mounting evidence that suggests BNP circulates in different structural forms that impact HF diagnosis and in vivo activity. Herein,we have developed and used an immunoaffinity purification assay to isolate endogenous BNP-32 from New York Heart Association class IV patient plasma for subsequent analysis by nano-liquid chromatography (LC) electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS. We have introduced stable isotope-labeled BNP-32 to the assayed plasma to enable quantification of endogenous levels of IBNP-32. Unlike the chemically nonspecific point-of-care tests (POCTs) and RIAs used worldwide to quantify BNP-32 from plasma, FT-ICR-MS (unprecedented mass measurement accuracy) coupled with LC (retention time) affords extraordinary molecular specificity, and when combined with the use of internal standards is able to confidently identify and quantify BNP-32. The significance of this work is despite exceedingly high circulating levels of BNP-32 in the New York Heart Association class IV patients as determined by POCTs (> 290 fmol/ml) nano-LC electrospray ionization-FT-ICR-MS data did not reveal any endogenous BNP-32. These results provide molecularly specific evidence for the absence of circulating BNP-32 in advanced-stage HF patients and suggest the existence of altered forms of BNP that are contributing to the POCT values.