title: Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure.
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 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.
authors.affiliation: Division of Clinical Biochemistry, Innsbruck Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Angelika
authors.initials: A
authors.lastname: Hammerer-Lercher
authors.forename: Bernhard
authors.initials: B
authors.lastname: Halfinger
authors.forename: Bettina
authors.initials: B
authors.lastname: Sarg
authors.forename: Johannes
authors.initials: J
authors.lastname: Mair
authors.forename: Bernd
authors.initials: B
authors.lastname: Puschendorf
authors.forename: Andrea
authors.initials: A
authors.lastname: Griesmacher
authors.forename: Norberto A
authors.initials: NA
authors.lastname: Guzman
authors.forename: Herbert H
authors.initials: HH
authors.lastname: Lindner
mesh_terms: D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods; D006031:Glycosylation; D006333:Heart Failure / Q000097:blood* / Q000503:physiopathology; D006801:Humans; D020097:Natriuretic Peptide, Brain / Q000097:blood*; D010446:Peptide Fragments / Q000097:blood*; D012720:Severity of Illness Index; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; C109794:pro-brain natriuretic peptide (1-76); D020097:Natriuretic Peptide, Brain