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

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction – current clinical problem 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: 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 questionsResearch 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 MethodsMaterials 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 -800C 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)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)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_technologies/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© 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 The antibody binding sites on the 6 fragments of glycosylated NT-proBNPglycosylated NT-proBNP

3637

O-glycosylation

44 48 53 58NH2 COOH

761

*

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Table 1Table 1

Table 1. NT-proBNP tryptic and semi-tryptic peptides identified after immunoprecipitation (IP) from plasma.

Position* Peptide m/z z mass

1-21 HPLGSPGSASDLETSGLQEQR.N 722.68 3 -0.003

3-21 P.LGSPGSASDLETSGLQEQR.N# 966.46 2 -0.004

4-21 L.GSPGSASDLETSGLQEQR.N 909.92 2 -0.005

7-21 P.GSASDLETSGLQEQR.N 789.36 2 -0.004

67-76 K.MVLYTLRAPR^ 407.23 3 0.001

67-75 K.MVLYTLRAP.R 532.30 2 0.002

67-73 K.MVLYTLR.A 448.25 2 0.001

*, amino acid position in mature NT-proBNP protein. #, non-tryptic

cleavages are bold. ^, missed cleavages are underlined.

© Copyright 2009 by the American Association for Clinical Chemistry© 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-Relative proportion of N- and C-terminal tryptic and semi-trypic peptides from NT-proBNP trypic peptides from NT-proBNP

© Copyright 2009 by the American Association for Clinical Chemistry© 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-proBNP13–76 concentration at the P < 0.05 level.

Circulating fragments of NT-proBNP in blood of HF patients Circulating fragments of NT-proBNP in blood of HF patients (n=20).(n=20).

© Copyright 2009 by the American Association for Clinical Chemistry© 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-proBNPCorrelation of NT-proBNP13-7613-76 to NT-proBNP to NT-proBNP1-20 1-20 and NT-and NT-

proBNPproBNP13-4513-45 in HF patients. in HF patients.C

© Copyright 2009 by the American Association for Clinical Chemistry

Key FindingsKey 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)

ConclusionNT-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.

© Copyright 2009 by the American Association for Clinical Chemistry

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