Received on April 13, 2007
Accepted on February 13, 2008

Proteomics and Protein Markers

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

¹ Division of Clinical Biochemistry, Innsbruck Biocenter, Innsbruck Medical University, Innsbruck, Austria; and Department of Medical and Chemical Laboratory Diagnostics, Innsbruck Medical University, Innsbruck, Austria
² Division of Clinical Biochemistry, Innsbruck Biocenter, Innsbruck Medical University, Innsbruck, Austria
³ Department of Internal Medicine, Clinical Division of Cardiology, Innsbruck Medical University, Innsbruck, Austria
⁴ Department of Medical and Chemical Laboratory Diagnostics, Innsbruck Medical University, Innsbruck, Austria

^* To whom correspondence should be addressed. 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.