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Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure

Hammerer-Lercher, Angelika and Halfinger, Bernhard and Sarg, Bettina and Mair, Johannes and Puschendorf, Bernd and Griesmacher, Andrea and Guzman, Norberto A. and Lindner, Herbert H. (2008) Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure. CLINICAL CHEMISTRY, 54 (5). pp. 858-865. ISSN Online ISSN: 1530-8561 Print ISSN: 0009-9147

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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.

Item Type:Article
Subjects:Biomedical Science > Nanomedicine
ID Code:593
Deposited By:M T V
Deposited On:10 Dec 2008 15:17
Last Modified:10 Dec 2008 15:17

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