Hammerer-Lercher Angelika, Halfinger Bernhard, Sarg Bettina, Mair Johannes, Puschendorf Bernd, Griesmacher Andrea, Guzman Norberto A, Lindner Herbert H
Division of Clinical Biochemistry, Innsbruck Biocenter, Innsbruck Medical University, Innsbruck, Austria.
Clin Chem. 2008 May;54(5):858-65. doi: 10.1373/clinchem.2007.090266. Epub 2008 Mar 13.
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).
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 MS 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.
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.
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.
人血液中出现的B型利钠肽原（proBNP）的具体形式尚不清楚。我们通过一种新的亲和色谱方法证明了人血浆中存在几种proBNP形式，该方法可与纳升液相色谱电喷雾电离串联质谱法（nano-LC-ESI-MS/MS）联合使用。
对于亲和色谱，我们将针对N端proBNP（NT-proBNP）表位1-21的多克隆羊抗体的Fab'片段偶联到硅胶珠上。我们将装有这些珠子的柱子（内径10 mm×0.8 mm）连接到胰蛋白酶反应器上，并使用预浓缩器与无 frit 纳喷柱相结合，对重度心力衰竭（HF）患者血浆的预提取和未提取样品中的proBNP形式进行质谱分析。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。
串联质谱实验证明，重度HF患者血浆的预提取样品中同时存在NT-proBNP和循环proBNP，蛋白质印迹分析显示有两条约23 kDa和13 kDa的条带，去糖基化后迁移到与重组proBNP和合成NT-proBNP位置相对应的位置。
我们获得了重度HF患者循环proBNP的确切证据，并首次证明了NT-proBNP的O-糖基化。蛋白质印迹分析中观察到的NT-proBNP和proBNP的分子量高于计算预期值，这可以通过这些肽在体内的O-糖基化来解释。
