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Clinical Chemistry 54: 858-865, 2008. First published March 13, 2008; 10.1373/clinchem.2007.090266
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(Clinical Chemistry. 2008;54:858-865.)
© 2008 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Analysis of Circulating Forms of proBNP and NT-proBNP in Patients with Severe Heart Failure

Angelika Hammerer-Lercher1,2,2, Bernhard Halfinger1,2, Bettina Sarg1, Johannes Mair3, Bernd Puschendorf1,3, Andrea Griesmacher2, Norberto A. Guzman1 and Herbert H. Lindner1,a

1 Division of Clinical Biochemistry, Innsbruck Biocenter, Innsbruck Medical University, Innsbruck, Austria; 2 Department of Medical and Chemical Laboratory Diagnostics, Innsbruck Medical University, Innsbruck, Austria; 3 Department of Internal Medicine, Clinical Division of Cardiology, Innsbruck Medical University, Innsbruck, Austria.

aAddress correspondence to this author at: Division of Clinical Biochemistry, Innsbruck, Biocenter, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. Fax 43-512-9003-73300; e-mail herbert.lindner{at}i-med.ac.at.

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 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.

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.






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