Circulating Fragments of N-Terminal ProB-Type

Natriuretic Peptides in Plasma of Heart Failure Patients

Jared Yong Yang Foo,
1
Yunxia Wan,
1
Benjamin L. Schulz,
2
Karam Kostner,
3,4
John Atherton,
3,5
Justin Cooper-White,
1,6
Goce Dimeski,
3,7
and Chamindie Punyadeera
1,6,8*
BACKGROUND: The use of nonstandardized N-terminal
proB-type natriuretic peptide (NT-proBNP) assays
can contribute to the misdiagnosis of heart failure
(HF). Moreover, there is yet to be established a com-
mon consensus regarding the circulating forms of NT-
proBNP being used in current assays. We aimed to
characterize and quantify the various forms of NT-
proBNP in the circulation of HF patients.
METHODS: Plasma samples were collected from HF pa-
tients (n 20) at rest and stored at 80 C. NT-
proBNP was enriched from HF patient plasma by use
of immunoprecipitation followed by mass spectromet-
ric analysis. Customized homogeneous sandwich Al-
phaLISA immunoassays were developed and vali-
dated to quantify 6 fragments of NT-proBNP.
RESULTS: Mass spectrometry identified the presence of
several N- and C-terminally processed forms of circu-
lating NT-proBNP, with physiological proteolysis
between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and
Pro75-Arg76. Consistent with this result, AlphaLISA
immunoassays demonstrated that antibodies targeting
the extreme N or C termini measured a low apparent
concentration of circulating NT-proBNP. The appar-
ent circulating NT-proBNP concentration was in-
creased with antibodies targeting nonglycosylated and
nonterminal epitopes (P 0.05).
CONCLUSIONS: In plasma collected from HF patients,
immunoreactive NT-proBNP was present as multiple
N- and C-terminally truncated fragments of the full
length NT-proBNP molecule. Immunodetection of
NT-proBNP was significantly improved with the use of
antibodies that did not target these terminal regions.
These findings support the development of a next gen-
eration NT-proBNP assay targeting nonterminal
epitopes as well as avoiding the central glycosylated
region of this molecule.
2013 American Association for Clinical Chemistry
Heart failure (HF)
9
is a global health problem, associ-
ated with poor clinical outcomes and substantial eco-
nomic burden to healthcare systems throughout the
world (1). Approximately 23 million people world-
wide live with HF, and this number is likely to increase
in the near future owing to an ageing and growing pop-
ulation (2). The measurement of either plasma B-type
natriuretic peptide (BNP) or N-terminal proBNP(NT-
proBNP) has been shown to improve diagnostic accu-
racy in patients suspected of HF (35) However, the
usefulness of these peptides for HF is limited by large
patient-to-patient variations, the presence of various
forms of the NT-proBNP peptide in blood (3), and
substantial differences in imprecision among detection
methods used. The latter becomes particularly relevant
when patients access different laboratory services that
use different diagnostic platforms (4, 5). In addition,
there is also no consensus about the circulating frag-
ments of NT-proBNP derived from the precursor
proBNP secreted fromthe heart tissue during HF. This
lack of consensus is further complicated by growing
evidence that the current NT-proBNP immunoassays
may detect blood proBNP in HF patients, which is
caused by the lack of processing of the precursor
proBNPby furin and corin protease convertase present
in the circulation (68).
To evaluate differences in analytical performance
and clinical results of BNP and NT-proBNP immunoas-
1
The Australian Institute for Bioengineering and Nanotechnology,
2
School of
Chemistry and Molecular Biosciences, and
3
School of Medicine, the University
of Queensland, Brisbane, Queensland, Australia;
4
Department of Cardiology,
Mater Adult Hospital, Brisbane, Queensland, Australia;
5
Department of Cardi-
ology, Royal Brisbane and Womens Hospital, Brisbane, Queensland, Australia;
6
School of Chemical Engineering, the University of Queensland, Brisbane, Queens-
land, Australia;
7
Chemical Pathology, Princess Alexandra Hospital, Brisbane,
Queensland, Australia;
8
current affiliation: Saliva Translational Research Group, The
University of Queensland Diamantina Institute, Woolloongabba, Australia.

Jared Yong Yang Foo, Yunxia Wan, and Benjamin L. Schulz contributed equally
to the work, and all should be considered as first authors.
* Address correspondence to this author at: Saliva Translational Research Group,
The University of Queensland Diamantina Institute, Level 6, TRI 37 Kent St. ,
Woolloongabba, QLD 4102, Australia. Fax 61-(0)7-3443-6966; e-mail
c.punyadeera@uq.edu.au.
Received November 26, 2012; accepted June 12, 2013.
Previously published online at DOI: 10.1373/clinchem.2012.200204
9
Nonstandard abbreviations: HF, heart failure; BNP, B-type natriuretic peptide;
NT-proBNP, N-terminal proBNP; NYHA, New York Heart Association; IP, immu-
noprecipitation; LC, liquid chromatography; MS/MS, tandem mass spectrometry;
LOD, limit of detection.
Clinical Chemistry 59:10
15231 (2013)
Proteomics and Protein Markers
1523
says, a proficiency testing program, called the Cardio-
OrmoCheck study, was carried out by Clerico et al. to
determine the measurement imprecision (8.7%) of
NT-proBNP concentrations using 3 current NT-
proBNP immunoassays that utilize antibodies and
standards fromRoche diagnostics (9). It is also evident
from the CardioOrmoCheck study that most of the
Italian laboratories used NT-proBNP in place of BNP.
Roche NT-proBNP assays utilize different antibodies
(polyclonal and monoclonal) targeting different
epitopes of NT-proBNP (10).
Ala-Kopsala et al. have demonstrated that there
are variants (truncations at both amino and carboxyl
termini) of circulating NT-proBNP in the plasma col-
lected from HF patients (11), giving rise to several im-
munoreactive NT-proBNP molecules smaller than the
full-length fragment of 76 amino acids. However, this
study did not focus on quantifying and identifying the
forms of circulating NT-proBNP in HF patients.
Quantification and reaching a consensus on the major
molecular forms of NT-proBNP in circulation is likely
to lead to an improvement in the accuracy of the NT-
proBNP assay in the diagnosis and the management of
HF patients.
The aims of this study were to identify and quan-
tify the major form of circulating NT-proBNP in
plasma collected from HF patients, information that
can be used to advise the development of next genera-
tion diagnostic assays.
Materials and Methods
PARTICIPANTS AND SAMPLE COLLECTION
This research was approved by the University of
Queensland Medical Ethical Institutional Board and
the Mater Hospital Medical Ethical Review Board. All
participants were 18 years of age and gave written
informed consent before donating samples for our
study. We recruited symptomatic HF patients [n 20;
New York Heart Association (NYHA) functional class
3] with a left ventricular ejection fraction 40%froma
general cardiology department. HF diagnosis was con-
firmed by the cardiologist from Mater Adult Hospital
according to the guidelines for the prevention, detec-
tion, and management of chronic HF in Australia (12).
All study participants were asked to refrain from exer-
cise 24 h before sample collection. The participants
were of European, African, and Asian descent and had
no symptoms of fever and/or respiratory tract infec-
tion. Blood samples were collected into EDTA tubes
(Greiner Vacuette; Greiner Bioone) to minimize in
vitro degradation of NT-proBNP and then immedi-
ately centrifuged at 500g at 4 C for 10 min. Samples
were divided into aliquots and stored at 80 C.
PURIFICATION AND MASS SPECTROMETRY CHARACTERIZATION
OF ENDOGENOUS NT-proBNP
To identify the major proteolytic products of NT-
proBNP in the circulation, plasma from HF patients
(n 4) was used for the immunoprecipitation (IP)
reactions. In brief, the NT-proBNP monoclonal anti-
body (targeting 1320 amino acids) was chemically
coupled to Dynabeads M-270 Epoxy (Invitrogen) us-
ing EDS-NHS [1-ethyl-3-(3-dimethylaminopropyl)-
carboimide and N-hydroxysuccinimide] chemistry ac-
cording to the manufacturers instructions.
Enriched plasma NT-proBNP was digested with
trypsin in 50 mmol/L Tris-HCl pH7.5 with 10 mmol/L
dithiothreitol at 37 C for 16 h, desalted using C18
ZipTips (Millipore), and analyzed by liquid chroma-
tography (LC)-electrospray ionizationtandem mass
spectrometry (MS/MS) using a Prominence nanoLC
system (Shimadzu) on a TripleTof 5600 mass spec-
trometer with a Nanospray III interface (ABSCIEX), as
previously described (13). Approximately 2 g peptide
was desalted on an Agilent C18 trap (300- pore size,
5-m particle size, 0.3-mm i.d. 5 mm) at a flow rate
of 30 L/min for 3 min, and then separated on a Vydac
EVEREST reversed-phase C18 HPLC column (300-
pore size, 5-m particle size, 150-m i.d. 150 mm)
at a flowrate of 1 L/min. Peptides were separated with
a gradient of 10%60%buffer B(80%acetonitrile with
0.1% formic acid) over 45 min, with buffer A (1% ace-
tonitrile and 0.1% formic acid) and buffer B. Gas and
voltage settings were adjusted as required. An MS TOF
scanfromanm/z of 350 to 1800 was performed for 0.5 s
followed by information-dependent acquisition of
MS/MS withautomatedcapillary electrophoresis selec-
tion of the top 20 peptides from m/z of 40 to 1800 for
0.05 s per spectrum.
Proteins were identified using Protein Pilot (AB
SCIEX), searching the LudwigNR database (down-
loaded from http://www.wehi.edu.au/faculty/
advanced_research_technologies/proteomics/wehi_
systems_biology_mascot_server as updated on 27
January 2012; 16 818 973 sequences; 5 891 363 821
residues) using the following standard settings: sam-
ple type, identification; cysteine alkylation, none; in-
strument, TripleTof 5600; species, no restriction; ID
focus, biological modifications; enzyme, trypsin;
search effort, thorough ID. False discovery rate anal-
ysis using ProteinPilot was performed on all
searches. Peptides identified with 99% confidence
and with a local false-discovery rate of 1% were
included for further analysis, and MS/MS fragmen-
tation spectra were manually inspected. Extracted
ion chromatograms were obtained using PeakView
1.1.
1524 Clinical Chemistry 59:10 (2013)
PLASMA NT-proBNP AlphaLISA IMMUNOASSAYS
We measured NT-proBNP immunoreactivity using 6
AlphaLISA immunoassays with different epitope spec-
ificities (Fig. 1A). All antibodies were of monoclonal
origin and were purchased from Hy Test (http://
www.hytest.fi) except for 11D1, which was from My
Biosource (MBS311067; http://www.mybiosource.
com). These monoclonal antibodies have been exten-
sively tested and validated for their specificity by the
manufacturers and have also been successfully used to
investigate the immunodetection of glycosylated NT-
proBNP circulating in human blood (14). Further-
more, these monoclonal antibodies have been exten-
sively tested for their specificity by Hy Test and My
Biosource in sandwich assays as capture and detection
antibodies for use with serum/plasma samples from
HF patients as well as for nonglycosylated recombi-
nantly expressed NT-proBNP and proBNP analytes.
The 6 NT-proBNP immunoassays are named
according to the first amino acid that the capture
antibody binds to on the NT-proBNP molecule and
the last amino acid that the detection antibody
binds to on the NT-proBNP molecule of 176
aminoacids: NT-proBNP
120
(5B6
112
and 13G12
1320
);
NT-proBNP
1345
(18H5
1320
and 11D1
2845
); NT-
proBNP
145
(5B6
112
and 11D1
2845
);
NT-proBNP
2876
(11D1
2845
and 28F8
6776
);
NT-proBNP
1376
(18H5
1320
and 28F8
6776
); NT-
proBNP
176
(5B6
112
and 28F8
6776
). Each NT-
proBNP AlphaLISA assay was prepared by using a pair
of monoclonal antibodies, with one antibody being bi-
otinylated and binding to the streptavidin-coated do-
nor beads (5B6, 11D1, or 18H5) and the second anti-
body being conjugated to the acceptor beads (11D1,
13G12, or 28F8) (15) (1619). The NT-proBNP ana-
lyte was purchased from PerkinElmer. The NT-
proBNP standards were prepared using pooled plasma
collected from healthy volunteers (n 10) and mixing
it with the High Block immunoassay buffer in a ratio of
50%:50%. This enabled us to overcome the matrix
O-glycosylaon
A
36 37 44 48 53 58
NH
2
COOH
76 1
*
B
120 AA
1345AA
145AA
2876 AA
1376 AA
176 AA
Fig. 1. Schematic diagram illustrating the antibody binding sites on the 6 fragments of glycosylated NT-proBNP and
MS peptide coverage of NT-proBNP enriched by immunoprecipitation from plasma.
(A), The 6 immunoassays use diagnostic-grade monoclonal antibodies to detect NT-proBNP
120
, NT-proBNP
1345
, NT-proBNP
145
,
NT-proBNP
2876
, NT-proBNP
1376
, and NT-proBNP
176
. N- and C-terminal proteolytic sites detected by our MS analysis are
shown with red vertical lines. *, Antibody pair that gave the highest apparent NT-proBNP concentration. (B). Sequences
corresponding to peptides identified with confidence 99% are bold. Nontryptic cleavage sites are shown with red vertical lines
(see Table 2).
Circulating NT-proBNP Fragments in HF Patient Plasma
Clinical Chemistry 59:10 (2013) 1525
effects that are commonly found when developing im-
munoassays in plasma.
BIOTINYLATION OF NT-proBNP MONOCLONAL ANTIBODIES
FOR IMMUNOASSAYS
N-hydroxysuccinimido-ChromaLink-biotin (2 g/L)
(9007105K, Solulink) was added to the respective
anti-NT-proBNPat a molar ratioof 30:1 andincubated
for 2 h at room temperature. Zeba spin desalting col-
umns (89882; Thermo Scientific Pierce) were used to
remove unbound biotin and purify the biotinylated
NT-proBNP antibody after incubation. The biotin-
ylated NT-proBNP antibody was stored at 4 C.
COUPLING OF NT-proBNP ANTIBODIES TO AlphaLISA ACCEPTOR
BEADS
Phosphate buffer solution(250 L) was addedto 50 L
of AlphaLISA acceptor beads (6772003; PerkinElmer)
and centrifuged at 16 000g for 15 min, and the super-
natant was discarded. To the acceptor bead pellet, 0.1
mg of NT-proBNP antibody, 1.25 L of 10% Tween-
20, 25 g of NaBH
3
CN, and PBS (0.13 mol/L, pH 7.4,
Gibco; Life Technologies) were added to make a final
reaction volume of 200 L, which was incubated for
24 h at 37 C. This was followed by the addition of 10
L of carboxy-methoxylamine to the reaction, and 1 h
of incubation at 37 C. The conjugated NT-proBNP
monoclonal antibody was purified by centrifugation at
16 000g for 15 min, the supernatant was discarded, and
the bead pellet was resuspended in 1 mL of 0.1 mol/L
Tris- HCl (pH 8). The purification step was repeated 3
times and was followed by sonication (20 pulses per
second). Conjugated beads were stored at 4 C.
IN-HOUSEDEVELOPED NT-proBNP AlphaLISA IMMUNOASSAYS
In brief, the AlphaLISA uses a pair of NT-proBNP an-
tibodies. One antibody is biotinylated and binds to the
streptavidin-coated donor beads while a second anti-
body is conjugated to the acceptor beads. In the pres-
ence of NT-proBNP, the beads come into close prox-
imity. The excitation of the donor beads promotes the
release of singlet oxygen molecules that triggers a cas-
cade of energy transfer to the acceptor beads, resulting
in a sharp peak of light emission at 615 nm (18).
Samples were analyzed in triplicates in 384-well
ProxiPlates
TM
(PerkinElmer). The only exception to
the manufacturer protocol was to reduce the total re-
action volume from 50 L to 10 L. In summary, the
assay consisted of a sample/analyte (1 L), biotinylated
antibody (25 mmol/L), and acceptor bead (25 ng/L)
mix, and streptavidin donor beads (80 ng/ L). For all
immunoassays, the end concentration of acceptor
beads was 10 g/mL, and the end concentration of bi-
otinylatedantibody was 1 nmol/L. The total incubation
time was 1.5 h at roomtemperature inthe dark, and the
plates were read on EnSpire plate reader.
ASSAY PERFORMANCE CHARACTERISTICS FOR NT-proBNP
AlphaLISA ASSAYS
To evaluate the suitability of the AlphaLISA assay for
measuring plasma NT-proBNP, we spiked 2 known
concentrations of NT-proBNP analyte in pooled
healthy control plasma. Both spiked and unspiked
samples were measured in the same AlphaLISA immu-
noassay. The recovery percentages of the 2 spiked
plasma samples were calculated in reference to
corresponding unspiked pooled plasma in a single
AlphaLISA, using the following equation (20):
Percentage recovery %
NT-proBNP]
spiked plasma
NT-proBNP]
unspiked plasma
/
NT-proBNP]
spiked
} 100.
Triplicates of plasma samples were run in a single
AlphaLISA assay and 3 independent AlphaLISA assays
(20). We assessed the limit of detection (LOD) for the
assays using 12 blanks (without NT-proBNP) in tripli-
cates in 1 run. The LOD was read from a sigmoidal
doseresponse curve based on the signal (21):
LOD signal count
average of blank signal count
3 SD of blank signal)].
STATISTICAL ANALYSIS
All statistical analyses were performed using GraphPad
Prism 5 software version 5.03 (GraphPad Software). A
standard curve was generated by plotting the raw
AlphaLISA counts vs the NT-proBNP standards using
a 4-parameter logistic equation (sigmoidal dose
response curve with variable slope) and a 1/y
2
data
weighting (minimizes relative distances squared). Be-
fore statistical analyses, the DAgostino and Pearson
omnibus normality test was performed on the plasma
NT-proBNP concentrations (continuous variables) of
the 6 fragments to test for normal distribution. To
compare values without normal distribution, a Wil-
coxon matched-pairs test was performed on data from
2 paired groups. Differences between 2 groups were
considered statistically significant at P 0.05. Spear-
man rank correlation coefficients were calculated to
investigate the relationship between 2 groups of con-
tinuous variables without normal distributions.
1526 Clinical Chemistry 59:10 (2013)
Results
We recruited HF patients at NYHA classification stage
3, and patient characteristics appear in Table 1.
Peptides from NT-proBNP were confirmed in the
elution fractions following IP of HF samples using an-
tibodies specific to NT-proBNP (Table 2). These pep-
tides covered the N- and C-terminal portions of the
predicted mature NT-proBNP protein (UniProtKBac-
cession P16860). However, several peptides were iden-
tified which were semitryptic peptides, in which one
end of the peptide was not the result of cleavage at a
consensus trypsin recognition site.
The relative abundance of these tryptic and semi-
tryptic peptides from NT-proBNP from each individ-
ual was determined using relative quantification of the
extracted ion chromatogram intensity of each peptide
form. The N- and C-terminal sets of peptides were in-
dividually normalized, providing a semiquantitative
measure of the abundance of N- and C-terminal cleav-
age fragments of NT-proBNP in the circulation of HF
patients. This showed that the relative proportions of
the differently processed forms of NT-proBNP were
essentially consistent between patients, and that circu-
lating NT-proBNP is subject to a high degree of both
N- and C-terminal proteolytic processing (Fig. 2).
We have performed a comparison (n 28) be-
tween the Roche diagnostic assay and our in-house
developed NT-proBNP
1376
(Spearman correlation of
r 0.69 and P 0.05) (see Fig. 1 in the Data Supple-
ment that accompanies the online versionof this article
at http://www.clinchem.org/content/vol59/issue10).
The NT-proBNP AlphaLISA assay performance is
summarized in Table 3.
In HF plasma samples, antibodies targeting the
full-length NT-proBNP as well as 5 antibody pairs tar-
geting different parts of the molecule were usedto mea-
sure NT-proBNP concentrations. The NT-proBNP
120
concentration ranged from 2182 to 19 808 ng/L, with a
median of 8885 ng/L (IQR, 416614 204 ng/L). The
NT-proBNP
145
concentrations ranged from 91.6 to
2645 ng/L, with a median of 448.3 ng/L (IQR, 195.2
860.3 ng/L). The NT-proBNP
1345
concentration
ranged from 165.1 to 14 164 ng/L, with a median
of 2151 ng/L (IQR, 840.53969 ng/L). The NT-
proBNP
1376
concentration ranged from 969.2 to
91 458 ng/L, with a median of 14 705 ng/L (IQR, 5045
28 999 ng/L). The NT-proBNP
2876
concentration
ranged from 140.8 to 2995 ng/L, with a median of 600.5
ng/L (IQR, 369.21339 ng/L). The NT-proBNP
176
con-
centration ranged from 399.7 to 16 091 ng/L, with a me-
Table 1. Characteristics of HF patients.
Parameter
HF patients
(n 20)
Age, years, mean (SD) 73 (10.9)
Sex, M:F, n 11:9
Body mass index, kg/m
2
, mean (SD) 29.9 (6.19)
NYHA classification All patients were
class 3
Systolic blood pressure, mmHg, mean (SD) 124 (3.81)
Diastolic blood pressure, mmHg, mean (SD) 75 (4.83)
Table 2. NT-proBNP tryptic and semitryptic
peptides identified after IP from plasma
Position
a
Peptide
b
m/z z mass
121 HPLGSPGSASDLETSGLQEQR.N 722.68 3 0.003
321 P.LGSPGSASDLETSGLQEQR.N 966.46 2 0.004
421 L.GSPGSASDLETSGLQEQR.N 909.92 2 0.005
721 P.GSASDLETSGLQEQR.N 789.36 2 0.004
6776 K.MVLYTLRAPR 407.23 3 0.001
6775 K.MVLYTLRAP.R 532.30 2 0.002
6773 K.MVLYTLR.A 448.25 2 0.001
a
Amino acid position in mature NT-proBNP protein.
b
Nontryptic cleavages are bold; missed cleavages are underlined.
Fig. 2. Relative proportion of N- and C-terminal tryp-
tic and semitrypic peptides from NT-proBNP purified
by IP from individual patients.
(A), N-terminal peptides: black, H1-R21; white, L3-R21;
gray, G4-R21; striped, G7-R21. (B), C-terminal peptides:
black, M67-R76; white, M67-P75.
Circulating NT-proBNP Fragments in HF Patient Plasma
Clinical Chemistry 59:10 (2013) 1527
dian of 4201 ng/L (IQR, 13708244 ng/L). The antibody
pair recognizing the NT-proBNP
1376
fragment gave the
highest concentration compared with the antibody pairs
recognizing the other parts of the molecule (P 0.05)
(Fig. 3A). The correlations betweenNT-proBNP
1376
and
the 5 fragments are shown in Fig. 3, BF.
Discussion
We have employed a combination of MS analysis
and AlphaLISA immunoassays to characterize the
circulating forms of NT-proBNP in HF plasma. The
6 in-housedeveloped AlphaLISA immunoassays
demonstrated good analytical sensitivities for the
quantification of NT-proBNP fragments. We con-
trolled for matrix effects by pooling healthy control
plasma (n 10) and by spiking with known concen-
trations of NT-proBNP analyte, and we obtained re-
coveries between 70.2% and 121%. These recoveries
are a good indication that the NT-proBNP immunoas-
says are suitable for use with plasma samples. These
assays were then used to quantify and compare differ-
ent fragments of NT-proBNP in HF patients plasma
and to identify the antibody pair that gave the highest
apparent concentration of NT-proBNP in circulation.
Our MS results identified several peptides which
were semitryptic peptides, in which one end of the pep-
tide was not the result of cleavage at a consensus trypsin
recognition site. These semitryptic peptides were
most likely due to physiological cleavage at the N and
C termini of circulating NT-proBNP before IP (22).
The identity of the protease or proteases responsible for
these cleavage events in NT-proBNP is not clear, nor is
whether the cleavage events occur before or after cleav-
age of proBNP to NT-proBNP and BNP. Our MS anal-
yses did not detect peptides from BNP, suggesting that
the identified peptides originated from NT-proBNP
rather than proBNP. However, it is possible that some
proBNP is present in samples after IP. No semitryptic
peptides were detected that could result from internal
cleavage, which suggested that these nontryptic cleav-
age sites were not due to processing artifacts, but rep-
resented distinct forms of circulating NT-proBNP with
various extents of proteolytic processing at the Nand C
termini (Fig. 1B). Furthermore, MS data indicated that
a substantial fraction of circulating NT-proBNP was
subjected to truncation at both Nand Ctermini (Table
2, Figs. 1 and 2). This suggested that antibody pairs
for measuring the concentration of circulating NT-
proBNP should ideally not target the extreme N or C
termini of the peptide. We tested if this was the case by
comparing the apparent NT-proBNP concentrations
using antibody pairs targeting different segments
of NT-proBNP. The antibody pair targeting NT-
proBNP
1376
gave a higher concentration than
NT-proBNP
176
, and the antibody pair targeting NT-
proBNP
1345
gave a higher concentration than NT-
proBNP
145
(Fig. 3). These results are consistent with
truncationat theNterminus of NT-proBNP(Table2, Fig.
1), limiting binding of monoclonal antibody 5B6
(112)
to
this section of the molecule, leading to a lower apparent
NT-proBNP concentration. Similarly, the antibody pair
recognizing NT-proBNP
120
gave a higher concentration
thanNT-proBNP
176
(Fig. 3), consistent withC-terminal
truncation(Table 2, Fig. 1), limiting binding of monoclo-
nal antibody 28F8
(6776)
. These findings confirm our MS
data that the majority of circulating NT-proBNP is trun-
cated at its N and C termini, and is also consistent with
previous descriptions of multiple forms of circulating
NT-proBNP (4, 11).
Table 3. Performance characteristics of our NT-proBNP immunoassays.
AlphaLISA immunoassay
Recovery, % 300 ng/L
% 3000 ng/L/,
Intraassay variation,
%
Interassay variation,
% (SE) LOD, ng/L
NT-proBNP
120
101.9 6.55 (0.88) 8.78 (0.56) 90.7
82.6
NT-proBNP
1345
81.0 9.14 (0.76) 6.69 (0.70) 52.4
80.0
NT-proBNP
145
77.8 7.58 (1.09) 7.13 (0.65) 26.4
76.0
NT-proBNP
2876
96.7 7.34 (0.62) 9.59 (1.03) 167.6
78.8
NT-proBNP
1376
88.1 7.30 (0.69) 6.32 (0.88) 147.3
121.0
NT-proBNP
176
71.5 5.39 (0.75) 4.46 (0.59) 45.3
70.2
1528 Clinical Chemistry 59:10 (2013)
The glycosylation of the central region of NT-
proBNP has also been reported to interfere with
immunodetection (14, 23). The apparent NT-proBNP
concentration using the antibody pair targeting NT-
proBNP
120
was higher thanwhenusing antibody pairs
targeting NT-proBNP
145
, NT-proBNP
1345
, or NT-
proBNP
2876
(Fig. 3). This is consistent with glycosyl-
ation at the central part of the NT-proBNP resulting in
weak binding of the antibodies to this region, corrob-
orating previous findings (7, 14, 23). Together, these
data suggest that an ideal immunoassay for NT-
proBNP should not target the extreme N- and
C-terminal regions of the NT-proBNP molecule and
should also avoid the central glycosylated region.
We previously validated our NT-proBNP
176
AlphaLISA by using 37 plasma samples that had also
been measured for NT-proBNP concentrations using
the Roche Diagnostic assay. A significant correlation
between our assay and the Roche assay (r
2
0.78 and
P 0.001) was observed (see online Supplemental Fig.
1) (15). The current second generation NT-proBNP
assay from Roche Diagnostics is based on 2 monoclo-
nal antibodies recognizing epitopes within amino acids
2731 and 4246 of NT-proBNP (Roche Diagnostics
data sheet; see http://www.aacc.org/publications/cln/
2008/July/Pages/newproducts7_0708.aspx). In con-
trast, in this study we have found that of the antibody
pairs we tested, the pair targeting NT-proNBP
1376
Fig. 3. Comparison and correlation of the concentrations of plasma NT-proBNP
1376
(the highest apparent concen-
tration) vs plasma NT-proBNP
120
, NT-proBNP
1345
, NT-proBNP
145
, NT-proBNP
2876
, and NT-proBNP
176
in HF
patients (n 20).
(A), The 25th, 50th (median), and 75th percentiles are indicated on the box-and-whisker plots. *, Significantly different from
NT-proBNP
1376
concentration at the P 0.05 level. Spearman rank correlation was calculated between the concentrations of
plasma NT-proBNP
1376
and (B) NT-proBNP
120
, Spearman r 0.890, P 0.0001; (C) NT-proBNP
1345
, Spearman r 0.859,
P 0.0001; (D) NT-proBNP
145
, Spearman r 0.788, P 0.0001; (E) NT-proBNP
2876
, Spearman r 0.908, P 0.0001;
and (F) NT-proBNP
176
, Spearman r 0.946, P 0.0001.
Circulating NT-proBNP Fragments in HF Patient Plasma
Clinical Chemistry 59:10 (2013) 1529
yielded the highest apparent concentration in plasma
samples collected from HF patients. However, our re-
sults suggestedthat anideal NT-proBNPimmunoassay
should not use an antibody targeting amino acids 67
76, due to C-terminal truncation of NT-proBNP (Figs.
1 and 2) reducing its apparent concentration (Fig. 3).
An alternative antibody targeting a section of NT-
proBNP between its central O-glycosylated region and
its truncated C terminus, but avoiding either region,
wouldbe preferable. However, no suchantibody is cur-
rently available. Additionally, antibodies targeting NT-
proBNP
1376
gave a relatively high interindividual vari-
ability. Hence it will be of interest to determine in a
large clinical study whether the detection of the NT-
proNBP
1376
fragment in plasma samples is preferable
to the current fragments detected by commercial diag-
nostic assays. A limitation to our study was that we
used a small cohort of HF patients classified as NYHA
functional class 3.
Recent work by Semenov et al. indicated that HF
patients tend to have an inefficient mechanism of con-
verting proBNP (precursor molecule) by furin conver-
tase into NT-proBNP and BNP upon secretion from
cardiomyocytes into the circulation (7). However, our
MS analysis did not identify proBNP fragments in the
circulation of HF patients (n 4), perhaps because of
the antibody that we chose for immunoprecipita-
tion. Katrukha et al. previously demonstrated that
the highly glycosylated region of plasma NT-
proBNP
2845
from HF patients was inaccessible to
site-specific antibodies directed at this region, and
this was further proven by the enzymatic removal of
O-glycosylated oligosaccharide molecules from
these regions that resulted in a significant (P 0.05)
increase in NT-proBNP concentrations post degly-
cosylation (24). Therefore, results from our study
paralleled previous findings, in which monoclonal
NT-proBNP
2845
antibodies were unable to bind to
the O-glycosylated regions of human NT-proBNP.
The relatively strong correlations of the concentra-
tions of plasma NT-proBNP
1376
(nonglycosylated
region) with NT-proBNP
1345
and NT-proBNP
2876
suggested that the concentrations of O-glycosylation
(nonglycosylated vs glycosylated) in endogenous
NT-proBNP are comparatively constant in a selected
group of chronic HF patients (Fig. 3, C and E). This
is supported by the work from Nishikimi et al., who
recently reported that the ratios of glycosylated and
nonglycosylated forms of plasma NT-proBNP are
constant in all HF patients (NYHA class 14) (25).
The presence of O-linked oligosaccharide mole-
cules on the Ser and Thr residues in the region of hu-
man NT-proBNP
2845
have been reported in the liter-
ature to maintain the stability of NT-proBNP in the
circulation (26). Therefore, monoclonal antibodies
that target the nonglycosylated sites between NT-
proBNP
2845
[i.e., NT-proBNP
3035
(Glu-Leu-Gln-
Val-Glu-Gln motif)] are likely to exclude the effects of
O-glycosylation on the measurement of plasma NT-
proBNP. This, together with monoclonal antibodies
targeting NT-proBNP
1320
, is likely to provide a more
standardized measurement of the plasma NT-proBNP
for future sandwich immunoassays. Our data indi-
cated that although immunoreactive NT-proBNP was
present in human plasma as multiple fragments of the
full-length NT-proBNP molecule, specific epitopes of
the peptide appear to be more abundant, providing
ideal targets for developing the next generation diag-
nostic assays for clinical use.
Insummary, we have providedevidence that anideal
immunoassay to detect NT-proBNP in plasma should
not target the extreme N- and C-terminal regions of NT-
proBNP, because of endogenous proteolytic truncations
at these sites, and should also not target the central
O-glycosylatedsectionof the protein. These results will be
important for the development of next generation NT-
proBNP immunoassays for diagnostic purposes. Our
findings will pave the way for the development of a more
standardized commercial third generation NT-proBNP
immunoassay to detect the presence of HF.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors Disclosures or Potential Conflicts of Interest: Upon man-
uscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: B.L. Schulz, National Health and Medical Re-
search Council Project Grant 631615 and National Health and Med-
ical Research Council Career Development Fellowship APP1031542;
C. Punyadeera, Queensland Government Smart Futures Fellowship
Programme (QGSFF), University of Queensland NewStaff Research
Funds (UQNSRSF 601252), University of Queensland Foundation
Research Excellence Award Scheme, and donations of NT-proBNP
monoclonal antibodies from Perkin Elmer (USA).
Expert Testimony: None declared.
Patents: None declared.
Role of Sponsor: The funding organizations played no role in the
designof study, choice of enrolledpatients, reviewandinterpretation
of data, or preparation or approval of manuscript.
Acknowledgments: The authors acknowledge the help of Fairuz Ja-
maluddin with the zip-tipping of 4 plasma samples from the HF
patients for MS analysis.
1530 Clinical Chemistry 59:10 (2013)
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Circulating NT-proBNP Fragments in HF Patient Plasma
Clinical Chemistry 59:10 (2013) 1531