Circulating Fragments of N- Terminal Pro B-Type Natriuretic Peptide in Plasma of Heart Failure Patients J.Y.Y. Foo, Y. Wan, B.L. Schulz, K. Kostner, J. Atherton, J. Cooper-White, G. Dimeski, and C. Punyadeera October © Copyright 2013 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Introduction – current clinical problem  Heart failure (HF):  a global health problem  associated with poor clinical outcomes  substantial economic burden throughout the world  Plasma B-type natriuretic peptide (BNP) or N-terminal proBNP (NTproBNP) improve diagnostic accuracy in patients suspected of HF  However, their utility in screening asymptomatic populations and for monitoring is limited by between and within patient variation, and the presence of various forms of the NT-proBNP peptide in blood

© Copyright 2009 by the American Association for Clinical Chemistry Aims of study:  identify and quantify the major form of circulating NT-proBNP in plasma collected from HF patients  inform the development of next generation diagnostic assays

© Copyright 2009 by the American Association for Clinical Chemistry Research questions  What are the common circulating fragments of NT- proBNP in blood of HF patients?  Is NT-proBNP in circulation (i.e. in blood) fragmented?  Which fragment should be the target of NT- proBNP assays for new diagnostic purposes?

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods  Participants and sample collection  All participants were >18 years of age and gave written consent  Recruited 20 symptomatic HF patients (New York Heart Association (NYHA) functional class III - IV)  Blood samples were collected into EDTA tubes to minimize in vitro degradation of NT-proBNP, immediately centrifuged, plasma separated and aliquots stored at C until analysed  Immunoprecipitation  Used to identify the major proteolytic products of NTproBNP in the circulation  Plasma from HF patients (n= 4) was used for the immunoprecipitation (IP) reactions

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (continued)  Immunoprecipitation and mass spectrometry  NT-proBNP monoclonal antibody (targeting amino acids 13–20 ) was chemically coupled to Dynabeads® M-270 Epoxy (Invitrogen) using EDS-NHS [1-ethyl-3-(3 dimethylaminopropyl)-carboimide and N-hydroxysuccinimide] chemistry according to the manufacturer’s instructions  Enriched plasma NT-proBNP was digested with trypsin in 50 mmol/L Tris-HCl pH 7.5 with 10 mmol/L dithiothreitol at 37°C for 16 h, desalted using C18 ZipTips (Millipore), and analyzed by liquid chromatography (LC)-electrospray ionization–tandem mass spectrometry (MS/MS) using a Prominence nanoLC system (Shimadzu) on a TripleTof 5600 mass spectrometer with a Nanospray III interface (AB SCIEX)

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (continued)  Immunoprecipitation and mass spectrometry  Proteins were identified using Protein Pilot (AB SCIEX), searching the LudwigNR database (downloaded from ogies/proteomics/wehi_systems_biology_mascot_server as updated on 27January 2012; 16,818,973 sequences; 5,891,363,821 residues)  Peptides identified with 99% confidence and with a local false-discovery rate of 1% were included for further analysis, and MS/MS fragmentation spectra were manually inspected  Extracted ion chromatograms were obtained using PeakView 1.1

© Copyright 2009 by the American Association for Clinical Chemistry Figure 1. The 6 immunoassays use diagnostic grade monoclonal antibodies to detect NT- proBNP1-20, NT-proBNP13-45, NT-proBNP1-45, NT-proBNP28-76, NT-proBNP13-76 and NT-proBNP1-76. N- and C-terminally proteolytic sites detected from our study are shown with red vertical lines. * Antibody pair that gives the highest NT-proBNP concentration. The antibody binding sites on the 6 fragments of glycosylated NT-proBNP O-glycosylation NH 2 COOH 761 *

© Copyright 2009 by the American Association for Clinical Chemistry Table 1 Table 1. NT-proBNP tryptic and semi-tryptic peptides identified after immunoprecipitation (IP) from plasma.

© Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Relative proportion of N- and C-terminal tryptic and semi-trypic peptides from NT-proBNP purified by IP from individual patients. (A) N-terminal peptides: blue, H1-R21; red, L3-R21; green, G4-R21; purple, G7-R21. (B) C-terminal peptides: orange, M67-R76; pink, M67-P75. Relative proportion of N- and C-terminal tryptic and semi- trypic peptides from NT-proBNP

© Copyright 2009 by the American Association for Clinical Chemistry Figure 3. The 25th, 50th (median), 75th percentiles are indicated on the box and whisker plots. * Significantly different from NT-proBNP 13–76 concentration at the P < 0.05 level. Circulating fragments of NT-proBNP in blood of HF patients (n=20).

© Copyright 2009 by the American Association for Clinical Chemistry Figure 4. Spearman’s rank correlation was performed between the levels of plasma NT-proBNP13-76 and NT-proBNP1-20 (Spearman’s r=0.890, p<0.0001) and NT- proBNP13-45 (Spearman’s r=0.859, p<0.0001). Correlation of NT-proBNP to NT-proBNP 1-20 and NT- proBNP in HF patients. C

© Copyright 2009 by the American Association for Clinical Chemistry Key Findings  Antibodies targeting the N-terminus give low apparent concentrations (1-76 vs 13-76; 1-45 vs 13-45).  Antibodies targeting the C-terminus give low apparent concentrations (1-76 vs 1-20).  Antibodies targeting the glycosylated region give low apparent concentrations (1-45 vs 1-76) Conclusion NT-proBNP is proteolytically truncated at both the N- and C- termini and is glycosylated in its central region. For an optimal immunoassay, antibodies should not target these regions.

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