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Characterization of molecular forms of N-terminal B-type natriuretic peptide in vitro.

The heart secretes A-type natriuretic peptides (ANPs) [3] and B-type natriuretic peptides (BNPs), which regulate body fluid homeostasis and vascular tone (1). Circulating concentrations of ANP and BNP, and their N-terminal counterparts (N-terminal profragment of ANP [NT-proANP] and N-terminal profragment of BNP [NT-proBNP]), are used as markers of heart failure and other cardiac disorders (2, 3). Analytical problems have been encountered with assays of circulating NT-proBNP, relating especially to assay specificity and analyte stability (4-7). Antisera are often raised against synthetic fragments of NT-proBNP without knowledge of how well they recognize the extended full-length peptide. Furthermore, there is no consensus about the exact circulating forms of peptides derived from proBNP (8-10). Schellenberger et al. (11) reported O-linked glycosylation of proBNP that hampers antibody recognition (12). We previously found molecular heterogeneity, not related to glycosylation, that has a major impact on the measurement of NT-proBNP (13, 14). To facilitate rational assay development, we wanted to further explore the nature and origin of the heterogeneity. The availability of recombinant peptides enabled us to focus on factors not related to the glycosylation. The results demonstrate that while there is major heterogeneity in circulating NT-proBNP, specific epitopes of the peptides are extraordinarily stable, providing ideal targets for clinically useful diagnostic assays.

Materials and Methods


We collected into vacuum tubes EDTA plasma (n = 8) and serum samples (n = 8) from healthy laboratory personnel. Dilated cardiomyopathy samples were from Kuopio University Hospital (15). We obtained informed consent from volunteers. The whole study is consistent with the Declaration of Helsinki.


Patient plasma samples with high concentrations of immunoreactive NT-proBNP were passed through a Sephadex G-75 Fine column (2.5 x 20 cm) and eluted with 300 mL/L acetic acid into 5-mL fractions. Immunoreactive fractions were pooled and lyophilized. Half of each sample was left as such and half was treated with trifluoromethanesulfonic acid (TFMS; Sigma Aldrich) according to established procedures (16). Excess reagents were then removed in a Sephadex G-15 column (1.5 x 30 cm) eluted with 300 mL/L acetic acid. The samples with and without the TFMS treatment were lyophilized and analyzed by reversed-phase HPLC (RP-HPLC) as described previously (13).


The coding region of human proBNP cDNA was amplified and cloned into pShuttle-CMV vector by using the primers 5'-GCG TCG ACT CCA GAG ACA TGG ATC CCC AG-3' (forward) and 5'-CCC AAG CTT TTA ATG CCG CCT CAG CAC-3' (reverse), and adenovirus was prepared by standard protocols as specified by the supplier of the vector (Qbiogene). The structure of the construct was verified by restriction enzyme digestion and sequencing. Rats were anesthetized with medetomidine hydrochloride and ketamine hydrochloride. The adenoviral construct (1 x [10.sup.9] infectious units in 100 [micro]L) was injected into the anterior wall of the left ventricle as previously reported (17). The animals were sacrificed 3 days after the gene transfer. Blood samples were collected into precooled tubes containing ethylenediamine tetra-acetic acid (1.5 g/L blood) and immediately centrifuged, and the plasma was stored at -80 [degrees]C. The hearts were weighed and the ventricles were immersed in liquid nitrogen and stored at -80 [degrees]C. Aliquots of left ventricle and plasma were extracted with Sep-Pak [C.sub.18] cartridges and analyzed by gel filtration HPLC (GF-HPLC) by using a Shimadzu Prominence LC system and Biosuite-125 HR7.8 x 300 mm column (Waters) eluted with 10% acetonitrile in aqueous trifluoroacetic acid (1 mL/L trifluoroacetic acid in water) at 1 mL/min. Fractions of 0.5 mL were collected and dried for use in the immunoassays. The experimental design was approved by the Institutional Animal Care and Use Committee of the University of Oulu.


Recombinant NT-[proBNP.sub.1-76] was prepared as reported previously (13). Blood samples were drawn, and 10-mL aliquots were immediately spiked with recombinant NT-[proBNP.sub.1-76] followed by separation of plasma or serum. A reference sample of assay buffer was similarly spiked with recombinant NT-[proBNP.sub.1-76]. The spiked serum, plasma, and assay buffer pools were divided into aliquots of 0.6 mL and stored at -20 [degrees]C, + 4[degrees]C, or room temperature for 0, 1, 2, 3, 4, and 8 weeks followed by RP-HPLC analysis. For reference purposes, patient samples with relatively high concentrations of immunoreactive NT-proBNP were also analyzed by RP-HPLC as described previously (13). The experiments were repeated with 3 patient samples with similar results. The spiked samples were run in conditions resulting in longer retention and thus better separation of the less hydrophobic components.


Plasma and serum were analyzed by reversed-phase HPLCbyusingaVydacC4 (4.6 [inner diameter] x 150 mm) column as previouslyreported (13, 14). The flow rate was 1 mL/min, and fractions of 0.5 or 1 mL were collected. NT-proBNP immunoreactive fractions (35-36, 47-48, 51-52), identified byRIAs, were further purified by GF-HPLC, each peak in separate runs. The samples were applied into a Protein-Pak GF-HPLC column (300 x 3.9 mm [inner diameter]; Waters) and eluted with 40% acetonitrile in aqueous trifluoroacetic acid (1 mL/L trifluoroacetic acid in water). The flow rate was 1 mL/min, and 0.4-mL fractions were collected. Immunoreactive peak fractions (20-22,20-21, 23-24; each 3 purified separately) were finally applied to a Vydac C4 column (4.6 [inner diameter] x 150 mm) and eluted with a linear 30-min gradient from 19.2% to 32% acetonitrile in aqueous trifluoroacetic acid (1 mL/L trifluoroacetic acid in water). The flow rate was 1 mL/min, and 0.5-mL fractions were collected. The N-terminal sequences of the peptides (fractions 26-27, 42, 44, 48) were identified by 6 cycles of automated Edman degradation on a Procise 492 protein sequencer (Applied Biosystems). The molecular masses were determined by mass spectrometry on a QToF I-ES mass spectrometer (Micromass).


Peptide concentrations were determined by RIAs specific to human NT-[proBNP.sub.1-22], NT-[proBNP.sub.10-29], NT-[proBNP.sub.57-76], and BNP-32 as previously reported (13, 14). The human NT-proBNP assays do not cross-react (<0.01%) with peptides derived from rat proBNP.


Recombinant NT-[proBNP.sub.1-76] (1 [micro]g) was dissolved in PBS (NaCl 140 mmol/L, KCl 2.7 mmol/L, [Na.sub.2]HP[O.sub.4] 10.1 mmol/L, K[H.sub.2]P[O.sub.4] 1.8 mmol/L, pH 7.4), fractionated in a 15% SDS-PAGE gel, and transferred onto a PVDF membrane. The membrane was blocked with nonfat milk (50 g/L), incubated with a-NT-[proBNP.sub.10-29] antiserum (1:5000) for 60 min at room temperature and washed with PBS-Tween-20 (1 mL/L). After incubation with the secondary a-Goat IgG-Peroxidase antibody (1:5000, Sigma Aldrich) for 60 min at room temperature, the membrane was washed and detected by chemifluorescence using Amersham ECL[TM] Plus Western Blotting Detection System (GE Healthcare).



The results are expressed as mean (SD). Data groups were analyzed by 2-way ANOVA followed by the Holm-Sidakposthoc test. A P value <0.05 was considered significant.



Immunoreactive NT-proBNP was analyzed by RP-HPLC and immunoassays specific to different epitopes of the peptide (Fig. 1). Unspiked patient plasma eluted in a single peak well before the recombinant NT-[proBNP.sub.1-76] calibrator (Fig. 1A). However, the broadness of the peak and the unequal reactivity with the different antisera suggests molecular heterogeneity. Treatment with TFMS (so-called chemical deglycosylation) altered the elution position, but a similar broad peak as in the untreated samples was detected (Fig. 1B). Interestingly, in our hands, TFMS treatment markedly diminished the amount of NT-proBNP immunoreactivity regardless of the epitope examined and destroyed NT-proANP immunoreactivity completely (data not shown), although the latter peptide is not glycosylated.

In the plasma samples obtained from healthyindividuals and spiked with at least a 10-fold higher amount of recombinant NT-[proBNP.sub.1-76], animmunoreactive peak was detected equally well by all 3 antisera when the samples were stored at -20 [degrees]C (Fig. 1C). In the serum samples, however, antiserum against the epitope NT-[proBNP.sub.10-29] detected markedly higher amount of immunoreactivity compared with the NT-[proBNP.sub.1-22] and NT-[proBNP.sub.57-76] antisera (Fig. 1D). After 1 week of storage at room temperature, 3 major immunoreactive NT-[proBNP.sub.10-29] components were detected in both plasma (Fig. 1E) and serum (Fig. 1F). The NT-[proBNP.sub.1-22] and NT-[proBNP.sub.57-76] antisera detected immunoreactivity in only the peak eluting late in the chromatogram but not in the earlier peaks.


To investigate the dynamics of the disappearance of NT-proBNP, whole blood was spiked with recombinant NT-proBNP. Aliquots of plasma and serum were then incubated for different periods of time at -20 [degrees]C, +4[degrees]C, or room temperature. Immunoreactive NT-proBNP was measured by assays specific to different epitopes of the peptide. The starting concentrations obtained with different assays were identical with the peptide incubated in assay buffer (Fig. 2, A and B). On the other hand, in plasma (Fig 2, C and D, P < 0.01) and especially in serum (Fig. 2, E and F, P < 0.001), immunoreactive NT-[proBNP.sub.1-22] concentration was clearly lower than that of NT-[proBNP.sub.10-29] or NT-[proBNP.sub.57-76], even at the start of the incubation, right after the serum was separated.


Only a fraction of the original amount of immunoreactive NT-[proBNP.sub.1-22] [24 (0.4)% in plasma, 22 (0.8)% in serum] or NT-[proBNP.sub.57-76] [14 (4.6)% in plasma, 28 (1.6)% in serum] was detectable after 1 week of storage of samples at room temperature, whereas the majority of immunoreactive NT-[proBNP.sub.10-29] was preserved [79 (4.5)% in plasma, 79 (2.1)% in serum] (Fig. 2, D and F). About half of the immunoreactive NT-[proBNP.sub.10-29] was still detectable at 8 weeks at room temperature [49 (2.7)% in plasma and 46 (2.6)% in serum]. At lower temperatures, the rate of decrease was slower (Fig. 2, C and E). The concentrations of all 3 different epitopes remained stable in samples kept in assay buffer for 4 weeks at +4[degrees]C (Fig. 2A) or 8 weeks at -20 [degrees]C (data not shown).


To identify the main products generated from recombinant NT-proBNP upon incubation, fresh blood samples were spiked with recombinant NT-proBNP, followed by separation of the plasma or serum and incubation at room temperature for 1 week. Four separate immunoreactive NT-[proBNP.sub.10-29] components were detected in both plasma and serum (serum in Fig. 3A, plasma data not shown), but the NT-[proBNP.sub.1-22] and NT-[proBNP.sub.57-76] assays recognized these peaks only weakly. The peaks designated "1," "2 + 3," and "4" were further separately purified to apparent homogeneity by GF-HPLC (Fig. 3B) and RP-HPLC with shallow gradient elution (Fig. 3C). N-terminal sequence analysis showed that all 4 peptides possess the intact N-terminal sequence of recombinant NT-proBNP (Table 1). On the basis of these data, the mass analyses (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www. were consistent with peak 1 corresponding to Gly-Ser-NT-[proBNP.sub.1-36] and peaks 2-4 to mixtures with varying proportions of Gly-Ser-NT-[proBNP.sub.1-62] and Gly-Ser-NT-[proBNP.sub.1-64] (Table 1). The N-terminal Gly-Ser dipeptide is derived from the cloning vector used for expression of the GSTNT-[proBNP.sub.1-76] fusion protein.



To find out whether the heterogeneity of circulating NT-proBNP is a characteristic of the peptide itself or brought upon by specific components in human plasma, we introduced by adenoviral transfer an expression vector overexpressing human proBNP to rat left ventricle. We then examined the molecular forms of immunoreactive human NT-proBNP in rat myocardium and plasma. The GF-HPLC fractions of myocardial cell lysates contained high-molecular weight human BNP and human NT-proBNP immunoreactive material (Fig. 4A) of a size consistent with human [proBNP.sub.1-108]. However, in plasma, there was immunoreactive material corresponding exactly to the size of the native circulating form human BNP-32, indicating that human proBNP can be processed correctly in rat cardiomyocytes. Immunoreactivity eluting at approximately the same position with the recombinant NT-[proBNP.sub.1-76] marker was detected by all 3 different NT-proBNP antisera. In addition, a major high-molecular weight component eluting at void volume was detected with the NT-[proBNP.sub.10-29] assay, but not the NT-[proBNP.sub.1-22], NT-[proBNP.sub.57-76], or BNP assays (Fig. 4B). Western analysis of recombinant human NT-proBNP revealed the rapid formation of at least 2 higher molecular forms, which according to their electrophoretic mobilities, correspond to a dimer and trimer of the monomeric NT-[proBNP.sub.1-76] peptide (Fig. 4C).



There is a large amount of molecular heterogeneity in circulating NT-proBNP, as demonstrated by previous studies (8, 11-14) and our present findings. O-linked glycosylation appears to be one source of the heterogeneity (11). We found that treatment with trifluoromethanesulfonic acid (chemical deglycosylation), while altering slightly the elution characteristics of circulating NT-proBNP, did not make the chromatographic profile of circulating immunoreactive NT-proBNP any less heterogeneous. The fact that the same treatment completely destroyed the immunoreactivity of circulating NT-proANP, which is not a glycoprotein, may cast some doubt on the specificity of the procedure. However, our main aim in the present study was to characterize the heterogeneity of NT-proBNP not related to glycosylation, byusing the recombinant peptides we had made previously (13).

Spiking blood samples drawn from healthy volunteers with a high concentration of recombinant human NT-[proBNP.sub.1-76] and incubating aliquots of plasma and serum at various temperatures allowed us to examine the time course of disappearance and changes in molecular forms of NT-proBNP. The 3 epitopes detected by our antisera (NT-[proBNP.sub.1-22], NT-[proBNP.sub.10-29], and NT-[proBNP.sub.57-76]) behaved very differently. The epitope NT-[proBNP.sub.10-29] was remarkably stable: approximately 80% of the original immunoreactivity remained in plasma and serum, even after incubation at room temperature, for 4 weeks. Immunoreactive NT-[proBNP.sub.1-22] and NT-[proBNP.sub.57-76] by contrast disappeared much faster: very little immunoreactivity was detectable after 1 week of storage at room temperature or 2 weeks at +4 [degrees]C. In fact, the preparation procedure for the serum samples byitselfresulted in the loss of about half of the NT-[proBNP.sub.1-22] immunoreactivity both at room temperature and at +4 [degrees]C. The very similar time course profiles and HPLC profiles obtained with EDTA plasma and serum samples (this study and that of Ala-Kopsala et al. (13)) suggest that EDTA-sensitive metalloproteinases do not have a major role in the degradation of NT-proBNP.

To examine the molecular details of the loss of immunoreactivity, whole blood from young healthy volunteers was spiked with a sufficiently high concentration of recombinant NT-[proBNP.sub.1-76] to allow isolation of the products. Serum and plasma samples were then incubated at room temperature for 1 week, and the major immunoreactive fragments formed were purified by HPLC. They were identified as Gly-Ser-NT-[proBNP.sub.1-62/64] and Gly-Ser-NT-[proBNP.sub.1-36] by N-terminal sequencing and mass spectrometry. Thus, the C-terminus was degraded as we had previously concluded by indirect evidence (13), but the presence of intact N-terminus was unexpected, given that the NT-[proBNP.sub.1-22] antiserum had difficulties in recognizing the peptides. Another puzzling finding was the HPLC behavior of the major NT-proBNP immunoreactive material, subsequentlyidentified as Gly-Ser-NT-[proBNP.sub.1-62] and Gly-Ser-NT-[proBNP.sub.1-64] (peaks 2-4 in Fig. 3). Despite their seemingly similar structures, both Gly-Ser-NT-[proBNP.sub.1-62] and Gly-Ser-NT-[proBNP.sub.1-64] were found in several resolved RP-HPLC peaks, indicating significant differences in their hydrophobicity. Differential glycosylation cannot explain these findings because we studied fragments derived from a recombinant peptide that does not contain polysaccharide side chains. Moreover, our antisera against NT-[proBNP.sub.1-22] and NT-[proBNP.sub.57-76] recognized only a few of the components, whereas all the components were equally well recognized by an antiserum against NT-[proBNP.sub.10-29]. Thus, it appears that there are structural modifications in the sequence between residues 1-15 and 26 -62 of NT-proBNP, which affect the hydrophobicity and immunoreactivity, but not the size, as determined by mass spectrometry.

A possible explanation for the divergent results obtained with different antisera could be that partial deamidation of asparagine and/or glutamine residues takes place in the degradation products in the acidic conditions used for chromatography. Deamidation decreases the hydrophobicityofpeptides, but a single deamidation increases the mass by only 1, a change that may go unnoticed in the mass spectrometric analysis. The recent evidence suggests that the recombinant NT-proBNP as well as the glycosylated form of proBNP are intrinsically unstructured proteins (18,19), making them susceptible to this kind of modification. However, the epitope sequence of anti-NT-[proBNP.sub.57-76] antiserum does not contain asparagine or glutamine residues, refuting the hypothesis of deamidation for this part of the peptide.

Another possibility for the detected heterogeneity could be oligomerization, as originally suggested by Seidler et al. (20). Reversible oligomerization is consistent with our present experimental findings with regard to the C-terminal half of NT-[proBNP.sub.1-62/64]. It could explain the chromatographic and immunologic heterogeneity without requiring size heterogeneity in the mass spectrometric analysis. Our proBNP gene transfer data lend support to this hypothesis. In rat plasma, a high-molecular weight component of immunoreactive human NT-proBNP was detected, which was not present in the corresponding myocardial cell lysates. Thus, it must have formed after secretion, probably in plasma. Moreover, this component was detected only by the NT-[proBNP.sub.10-29] assay, but not the NT-[proBNP.sub.1-22], NT-[proBNP.sub.57-76], or BNP ([proBNP.sub.77-108]) assays. The finding of oligomeric bands (di- and trimers) in the Western analysis of recombinant NT-[proBNP.sub.1-76] (Fig. 4C) supports the hypothesis of oligomerization as a source of circulating NT-proBNP heterogeneity.

The above results were obtained with exogenous recombinant NT-proBNP peptide. How well do the findings reflect the endogenous circulating NT-proBNP? The RP-HPLC profiles obtained with incubation of recombinant NT-proBNP in serum or plasma are similar to those obtained with endogenous circulating NT-proBNP (13). Moreover, despite the fact that the antisera we used (epitopes 1-22, 10-29, and 57-76) are not directed to the central sequence of NT-proBNP (amino acids 27-56) that contains the glycosylation sites, theyrecognized the endogenous circulating material with dissimilar affinity. These findings are not easily explainable on the basis of glycosylation, but instead indicate the occurrence of modifications not related to glycosylation at or near both termini of NT-proBNP. Our recombinant NT-proBNP has an N-terminal Gly-Ser-tag derived from the fusion protein used to manufacture it. We cannot rule out that the tag may interfere with N-terminal degradation of NT-proBNP, since it has been reported to be susceptible to the action of dipeptidyl dipeptidase (21). Nevertheless, the fact that very similar heterogeneity and epitope profiles have been found with endogeneous circulating NT-proBNP (13) and recombinant NT-proBNP (this study) argues that the minute structural difference does not affect the key conclusions of the present study. Thus, the results with endogenous peptide are in agreement with the recombinant peptide experiments.

Many of the NT-proBNP immunoassays currently used are based on antibodies directed against the terminal parts of NT-proBNP (9, 22-26). According to our present results, the N-terminus of NT-proBNP is vulnerable in blood to modifications that alter its immunoreactivity. Such modifications could explain the large variance of circulating NT-proBNP concentrations reported by different assays directed to the N-terminus (4, 6, 27, 28). In addition, C-terminus is susceptible to proteolysis, and the epitopes of the mid-fragment can be concealed by glycosylation (12). Therefore, special attention should be paid to epitope profiling in the design of NT-proBNP assays. In our experience, use of the epitope NT-[proBNP.sub.10-29] results in extraordinarily robust immunoassays. Since BNP is predominantly regulated at the level of transcription and mRNA stabilization (2, 3, 29), an assay that can detect the majority of the secreted peptide forms, regardless of their posttranslational modifications, should best reflect the actual regulation and state of the BNP system.

In this study, we found that NT-proBNP is susceptible in blood to modifications that interfere with immunoassays. The modifications of NT-proBNP include proteolysis, but also changes not relating to the peptide backbone, probably oligomerization. Our present findings help the development of robust immunoassays with better clinical usability for circulating natriuretic peptides.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (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 manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: M. Ala-Kopsala, Research Foundation of Instrumentarium; J. Rysa, Finnish Foundation for Cardiovascular Research and Ida Montin Foundation; H. Ruskoaho, Sigrid Juselius Foundation, Finnish Foundation for Cardiovascular Research, and Academy of Finland (Center of Excellence); O. Vuolteenaho, Sigrid Juselius Foundation, Finnish Foundation for Cardiovascular Research, and Academy of Finland.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Helka Koisti and Sirpa Rutanen for expert technical assistance.


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Minna Ala-Kopsala, [1] Anne-Mari Moilanen, [2] Jaana Rysa, [2] Heikki Ruskoaho, [2] and Olli Vuolteenaho [1] *

[1] Department of Physiology and [2] Department of Pharmacology and Toxicology, Institute of Biomedicine, Biocenter Oulu, University of Oulu, Oulu, Finland.

[3] Nonstandard abbreviations: ANP, A-type natriuretic peptide; BNP, B-type natriuretic peptide; NT-proANP, N-terminal profragment of ANP; NT-proBNP, N-terminal profragment of BNP; TFMS, trifluoromethanesulfonic acid; RP-HPLC, reversed-phase HPLC; GF-HPLC, gel filtration HPLC.

* Address correspondence to this author at: Department of Physiology, Institute of Biomedicine, P.O. Box 5000, FIN-90014 University of Oulu, Finland. Fax +358-8-5375320; e-mail

Received April 14, 2010; accepted September 1, 2010.

Previously published online at DOI: 10.1373/clinchem.2010.148775
Table 1. Major immunoreactive products formed from
recombinant NT-proBNP after incubation in serum at
room temperature. (a)

Peak Observed Observed mass
number N-terminal

1 GSHPLGSP 4018.08 [+ or -] 0.58

2 GS(-)PL 7117.69 [+ or -] 2.93

3 GSHPLG 6923.32 [+ or -] 0.89

4 GSHPLGSP 6923.33 [+ or -] 1.07
 ([approximately equal

 7115.70 [+ or -] 5.92
 ([approximately equal

Peak Proposed Expected Difference
number identity mass

1 4016.94 +1.14
2 .sub.1-36] 7116.85 +0.84

3 [GS-NT-proBNP 6922.62 +0.70
4 6922.62 +0.71
 .sub.1-62] 7116.85 -1.15



(a) The structures were determined by N-terminal
sequencing and mass spectrometry.
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Title Annotation:Proteomics and Protein Markers
Author:Ala-Kopsala, Minna; Moilanen, Anne-Mari; Rysa, Jaana; Ruskoaho, Heikki; Vuolteenaho, Olli
Publication:Clinical Chemistry
Date:Dec 1, 2010
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