In:
Clinical Chemistry, Oxford University Press (OUP), Vol. 54, No. 5 ( 2008-05-01), p. 858-865
Abstract:
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 × 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.
Type of Medium:
Online Resource
ISSN:
0009-9147
,
1530-8561
DOI:
10.1373/clinchem.2007.090266
Language:
English
Publisher:
Oxford University Press (OUP)
Publication Date:
2008
