title: Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro-B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure.
abstract: BACKGROUND
  Currently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms.
  
  
  METHODS
  The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry.
  
  
  RESULTS
  We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable.
  
  
  CONCLUSIONS
  Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms.
authors.affiliation: Division of Clinical Biochemistry and Protein Micro-Analysis Facility, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Halfinger
authors.affiliation: Institute of Laboratory Medicine, Kantonsspital Aarau AG, Aarau, Switzerland.
authors.forename: Angelika
authors.initials: A
authors.lastname: Hammerer-Lercher
authors.affiliation: Division of Clinical Biochemistry and Protein Micro-Analysis Facility, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Benno
authors.initials: B
authors.lastname: Amplatz
authors.affiliation: Division of Clinical Biochemistry and Protein Micro-Analysis Facility, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Sarg
authors.affiliation: Division of Clinical Biochemistry and Protein Micro-Analysis Facility, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Leopold
authors.initials: L
authors.lastname: Kremser
authors.affiliation: Division of Clinical Biochemistry and Protein Micro-Analysis Facility, Biocenter, Innsbruck Medical University, Innsbruck, Austria; herbert.lindner@i-med.ac.at.
authors.forename: Herbert H
authors.initials: HH
authors.lastname: Lindner
mesh_terms: D006031:Glycosylation; D006333:Heart Failure / Q000097:blood* / Q000175:diagnosis / Q000378:metabolism; D006801:Humans; D020097:Natriuretic Peptide, Brain / Q000097:blood* / Q000378:metabolism
chemical_list: D020097:Natriuretic Peptide, Brain