Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure
Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure
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DOI：
10.1373/clinchem.2007.090266
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发表时间：
2008-05-01
影响因子：
9.3
通讯作者：
Lindner, Herbert H.
中科院分区：
文献类型：
--
作者：
Hammerer-Lercher, Angelika;Halfinger, Bernhard;Lindner, Herbert H.
背景：人血液中B型利钠肽前体（proBNP）的具体形式尚不清楚。我们用一种新的亲和色谱方法证明了人血浆中存在几种proBNP形式，该方法可与纳米液相色谱电喷雾电离串联质谱（nano-LC-ESI-MS/MS）结合使用。方法：对于亲和色谱，我们将N-末端proBNP（NT-proBNP）表位1-21特异性的多克隆羊抗体Fab'片段偶联到硅胶珠上。我们将填充有这些珠粒的柱（10 mm. X 0.8 mm内径）连接到胰蛋白酶反应器，并使用预浓缩器与无熔块纳米喷雾柱组合来对来自患有严重心力衰竭（HF）的患者的血浆的预提取和非预提取样品中的proBNP形式进行NIS分析。我们在去糖基化实验中使用蛋白质印迹法来确认proBNP和NT-proBNP质量的变化。串联MS实验证明了在来自严重HF患者的血浆的预提取样品中存在NT-proBNP和循环proBNP，Western印迹分析显示约23 kDa和13 kDa的2条带，其在去糖基化后移动到对应于重组蛋白的位置。结论：我们获得了严重HF患者循环proBNP的明确证据，并首次证明了NT-proBNP的O-糖基化。在Western印迹分析中观察到的NT-proBNP和proBNP的分子量高于从计算中预期的分子量，这可以通过这些肽在体内的O-糖基化来解释。(c)2008年美国临床化学协会。
BACKGROUND: The specific forms of pro-B-type natriuretic peptide (proBNP) that occur in human blood are not yet clear. We demonstrated the presence of several proBNP forms in human plasma with a new affinity chromatography method that can be used in combination with nano-liquid chromatography electrospray ionization tandem mass Spectrometry (nano-LC-ESI-MS/MS).METHODS: For affinity chromatography, we coupled Fab' fragments of polyclonal sheep antibodies specific for N-terminal proBNP (NT-proBNP) epitope 1-21 to silica beads. We connected a column (10 mm. X 0.8 mm inner diameter) packed with these beads to a trypsin reactor and used a preconcentrator in combination with a fritless nanospray column to perform NIS analyses of proBNP forms in preextracted and non-preextracted samples of plasma from patients with severe heart failure (HF). We used Western blotting in deglycosylation experiments to confirm the shifts in proBNP and NT-proBNP masses.RESULTS: Tandem MS experiments demonstrated the presence of both NT-proBNP and circulating proBNP in preextracted samples of plasma from patients with severe HF, and Western blotting analyses revealed 2 bands of approximately 23 kDa and 13 kDa that shifted after deglycosylation to positions that corresponded to the locations of recombinant proBNP and synthetic NT-proBNP.CONCLUSIONS: We obtained clear evidence for circulating proBNP in patients with severe HF and provided the first demonstration of O-glycosylation of NT-proBNP. The higher molecular masses for NT-proBNP and proBNP observed in the Western blotting analyses than those expected from calculations can be explained by O-glycosylation of these peptides in vivo. (c) 2008 American Association for Clinical Chemistry.