1. 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 2013 www.clinchem.org/content/59/10/1523.full © Copyright 2013 by the American Association for Clinical Chemistry

2. © 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

3. © 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

4. © 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?

5. © 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 -80 0 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

6. © 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)

7. © 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 http://www.wehi.edu.au/faculty/advanced_research_technol 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

8. © 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 36 37 O-glycosylation 44 48 53 58 NH 2 COOH 761 *

9. © 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.

10. © 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

11. © 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).

12. © 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 13-76 to NT-proBNP 1-20 and NT- proBNP 13-45 in HF patients. C

13. © 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.

14. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Download the free Clinical Chemistry app on iTunes for additional content! Follow us