B-type natriuretic peptide (BNP) is a member of the natriuretic peptide (NP) family , . As an indicator for myocardial function evaluation, BNP has been applied in the clinical diagnosis of heart failure (HF) and acute myocardial infarction and in monitoring disease progression , , . In addition, drugs designed based on the amino acid structure and physiological mechanism of BNP have been proven to effectively relieve and even treat HF disease , , . BNP precursor and related peptides are also relevant for HF evaluation . The formation of the peptide is presented in Figure 1 . The European Society of Cardiology (ESC) defined the upper limit of normal in the non-acute setting as 125 ng/L BNP and 35 ng/L NT-proBNP . However, in infants, the concentrations of BNP and NT-proBNP are partly dependent on age and sex , .
The accurate measurement of NPs is no doubt the most significant event for patient diagnosis and treatment. However, the results of current clinical measurement are sometimes confusing. For instance, the BNP concentration of HF patients under the New York Heart Association classification IV (NYHA-IV) is defined as higher than 290 fmol/mL (≈1004 ng/L). Surprisingly, however, Hawkridge et al.  found very little BNP when using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Similarly, Niederkofler et al.  found a BNP range from 900 to 5000 ng/L by Biosite assay, but parallel measurements based on the MS method indicated an intact BNP concentration of only 25–43 ng/L in the circulation of HF patients. Miller et al.  developed a quantitative MS method for BNP metabolites and corin enzyme. The results of the Beckman assay and the developed MS approach exhibited poor correlation. These studies suggested that the results measured by immunoassays and MS approaches were quite different. In addition, for immunoassays, deviation was observed between different platforms . Saenger et al.  investigated five prevailing BNP immunoassay platforms and found that all assays exhibited substantial cross-reactivity with the proBNP peptide. In addition, from 2005 to 2011, more than 100 Italian laboratories participated in a BNP proficiency testing program, and the statistical data showed a 43.0% coefficient of variation in the BNP immunoassay . Franzini et al.  investigated the systematic differences between a new commercial method (ST-AIA-PACK in brief) and the Triage Biosite assay (a routine method used in reference laboratories) and found a 46.9% mean bias between them. Other similar cases were also reported in remarkable reviews , , . More recently, Vasile and Jaffe  examined the analytical characteristics of the seven most popular commercial BNP immunoassays and discussed the inherent problems.
The poor harmonization in BNP testing was caused by many factors, such as heterogeneity in the analytical methods, systematic error in immunoassays and quality attributes of the diagnostic reagents. Because BNP standardization has not been carried out or is still in the early stages worldwide, it has been difficult to elucidate the problem until now . However, cross-reaction is an undisputed clinical problem , , , . To explore these problems, this review proposes new materials and methods, including the development of International System of Units (SI)-traceable reference materials and reagents and the exploration of novel MS analytical methods.
Formation of BNP
The overloading of volume and pressure in the heart causes cardiac wall tension and stretching, volume overload, or ischemia, resulting in the increased production and release of NPs. After NPs are expressed and released, the precursor of proBNP is digested by enzymes such as corin and furin in circulation, generating equimolar amounts of the amino-terminal and carboxyterminal products, commonly referred to as NT-proBNP and BNP , , . Consequently, such peptides normally coexist in circulation , which foreshadows questions in BNP clinical measurement.
Metabolism and forms of BNP
BNP has a ring structure formed by cysteines. Previous studies have shown that BNP bioactivity is lost after ring structure disruption, which is commonly mediated by neutral endopeptidases in circulation . This structure plays a vital role in natriuretic peptide receptor (NPR) recognition . The combination of BNP and NPR-A could enhance the cyclic guanosine monophosphate (cGMP) pathway, leading to a series of physiological responses , . NPR-C plays a role in BNP clearance. He et al.  determined the crystal structure of the BNP/NPR-C complex.
In circulation, enzymes such as neprilysin, dipeptidyl peptidase IV (DPP-IV), insulin degrading enzyme and meprin will hydrolyze BNP at specific amino acids, generating truncated BNPs such as BNP 3-32, BNP 4-32 and BNP 5-32 , , . After enzymatic reactions, the immunological activity of BNP would be altered or even lost , , which is the reason the half-life of BNP in plasma is only approximately 22 min long . Obviously, the inhibition of these enzymes could maintain BNP bioactivity . Non-bioactive NT-proBNP exhibited better stability and could exist in plasma for approximately 7 days at room temperature . In contrast to proBNP, BNP is not modified by glycosylation . Seferian et al.  raised antibodies against a variety of forms of BNP and found that no posttranslational modification of these peptides occurred.
Because BNP and its metabolites might reflect different cardiac diseases or different degrees of myocardial injury, scientists seek to evaluate their bioactivity individually. Therefore, the development of biological methods is feasible but not sufficient. Chemical methods, such as chromatographic and mass spectrometric approaches, are effective for structure and quantity analysis. Therefore, the combination of biological and chemical approaches is a robust approach to such in-depth research.
To date, a variety of immune platforms and point-of-care testing products have been adopted for clinical BNP measurements. In addition, chromatographically assisted MS analytical methods such as high performance liquid chromatography coupled MS or capillary electrophoresis coupled MS (CE-MS) are emerging as reliable tools for the qualitative and quantitative measurement of BNP.
Clinical BNP assays
Worldwide, in vitro diagnostic (IVD) reagents and immunoassays have dominated the clinical testing laboratories. Because Access (Beckman Coulter) and Triage (Alere) did not characterize capture or detect epitopes, only five tests were performed here. The data in Table 1 and Figure 2 are from the IFCC website (http://www.ifcc.org/media/102208/NP%20Assay%20Table%20C%20SMCD%20vJuly_2011.pdf) and the reviews of Maalouf and Bailey  and Vasile and Jaffe . Commercial assays are sandwich-type assays based on two monoclonal antibodies or a combination of monoclonal and polyclonal antibodies . BNP 3-32, BNP 4-32, BNP 5-32 and even proBNP are also targets during BNP 1-32 assays.
Cross-reactivity of BNP
Before discussing cross-reaction, whether truncated BNPs and/or proBNP is the main form in HF patients is an important issue that needs to be investigated. Some studies have focused on this question and provided clear evidence. For instance, according to western blot results using antibodies directed against BNP, Liang et al.  reported that proBNP is a major form of immunoreactive BNP in HF plasma, which is similar to the results of Seferian et al. . For bioactivity analysis, one approach is to measure the second messenger cGMP, which was generated after NPR recognition . Based on this approach, Heublein et al.  showed that the stimulation of cGMP production by the DPP-IV product BNP 3-32 is similar to that of BNP 1-32 when tested on human cardiac fibroblasts or cardiomyocytes. Recently, by using immunocapture-assisted time-of-flight MS (TOF MS), Suzuki et al.  found that BNP 5-32 presented prognostic ability in HF patients. In addition, BNP 432, BNP 1-25, BNP 1-26 and even BNP 5-31 also retained bioactivity, giving cGMP responses similar to that of BNP 1-32 , . Therefore, one explanation for the findings of Adam and Eric is that the immunoassay results reflect the proBNP or truncated BNP concentrations instead of BNP 1-32, and the MS measurement results proved that only a small amount of BNP 1-32 existed in the sample , .
To eliminate cross-reactions with proBNP, Lewis et al.  designed a new polyclonal antibody that could recognize the amino-terminal sequence of BNP. The combination of homemade antibody and 50E1 was used in the BNP 1-32 immunoassay. Because such a combination could recognize both the amino- and the carboxyterminal of BNP 1-32, it would provide improved accuracy. As expected, compared with the combination of 24C5 and 50E1, the new method reduces cross-reactions with proBNP and truncated BNPs effectively at a concentration level of 5 nmol/L (≈1.73×104 ng/L), especially for BNP 3-32 and BNP 7-32 . These results are attractive, but the most current studies highlighted that the clinical BNP immunoassays in current use are far from complete.
Because BNP metabolites and proBNP are partly representative of BNP 1-32 in immunoassays, another solution for clinical use is to separate them before the assay. However, the separation of similarly structured NPs is complicated and time consuming, which does not meet the needs of clinical testing. Therefore, it seems that the development of a higher-specificity antibody is a more realistic and promising approach, similar to the encouraging work of Lewis et al. .
MS analytical methods
The MS platform generally includes separation devices, such as liquid chromatography (LC), ion exchange chromatography or capillary electrophoresis (CE) instruments. The superior resolution and accuracy of MS are provided by a mass analyzer. MS can provide an in-depth view of similar peptides because it can measure their mass-to-charge ratios (m/z). In combination with a high-resolution mass analyzer, such as TOF, Orbitrap or FT-ICR, the amino acids can be sequenced by the application of a matching algorithm to their fragment ions.
Because the abundant proteins in serum and plasma could inhibit MS intensity, cleanup is always a necessary operation. Solid-phase extraction (SPE) is the conventional cleanup method. Commercial SPE products can exclude albumin, IgG and IgA efficiently. Moreover, if any specific affinitive ligands are introduced on the SPE material, such as antibody-coupled beads or matrix, the resulting online chromatographic analysis is more sensitive .
As shown in Table 2, various platforms have been used to investigate the metabolic mechanism and the structure of intact BNP or truncated BNPs from patients. In practice, an additional offline cleanup procedure was adopted when using matrix-assisted laser desorption ionization (MALDI). Online LC or CE separation methods are commonly adopted in electrospray ionization sources (ESIs). The structural information obtained from MS methods is usually the key evidence to support and verify the hypothesis of the authors, and corresponding information is unavailable from the immunoassay approach . For instance, the signal peptide discussed in the work of Siriwardena et al.  was derived from pre-proBNP, although it does not belong to the BNP family. This approach provides a reference for further BNP studies. Fujimoto et al.  developed an immunocapture-assisted MALDI-TOF MS method to investigate whether truncated BNP could be indicative of ischemic heart disease, and they found that BNP 5-32 may reflect the pathophysiological process involved in restenosis.
Compared with identification studies performed by MS, quantitative MS studies are usually complicated. The cleanup recovery, quantitative range, limit of quantitation, method accuracy, precision and repeatability need to be investigated. According to the research purpose, samples could be analyzed on the intact, enzymatic peptide and/or amino acid level. Intact analysis is rapid, requires less sample pretreatment and is compatible with online operation; however, the intact standard is difficult to obtain. Peptide and amino acid analysis are more complex, but reference peptides or reference amino acids are easy to obtain. In addition, stable isotope-labeled peptides or amino acids are suitable for the evaluation of cleanup efficiency and even for absolute quantification. Parameters such as quantitative range, limit of quantitation, method precision and repeatability are closely related to the MS analyzer type and settings. Quantification accuracy is usually dependent on the quality and uncertainty of internal and external standards. Torma et al.  developed a method of SI-traceable quantification for reference material preparation. During this work, synthetic BNP was quantified at the amino acid and peptide levels, which were obtained from hydrolyzed by hydrochloric acid or enzymatically digested by trypsin. In addition, peptides labeled with heavy isotopic carbon atoms and nitrogen atoms were quantified based on the SI-traceable amino acids and then adopted as the internal standard. Each approach in Table 3 is designed to reduce matrix complexity before quantification, and immune purification and LC methods have become standard operations.
A summary of the immunoassay and MS analytical methods
MS-based methods are widely adopted in discovery research and are suitable for obtaining more accurate prediction and HF diagnosis . Although chromatographic approaches are time consuming and high in cost, the results are usually in depth and reliable . Therefore, such methods are more promising for BNP structure and activity investigation than for clinical application unless analytical cost reduction and efficiency improvement are realized. In clinical use, microbial identification by MALDI-TOF and neonatal disease screening are best examples to date . LC-MS and CE-MS approaches have become much more robust and convenient than before, but there is room for considerable further progress. At present, the development of a plasma BNP reference material using MS methods for use in immunoassays would be a comprehensive solution to harmonize the clinical results.
In immunoassays, antibodies are used for quantitation. Because BNP immunoassays commonly have poor specificity and the results cannot be traced to SI units, the immunoassay results are questionable. However, in MS methods, antibodies are tools used for biological sample cleanup; furthermore, the quantitative results are dependent on the standards spiked at the beginning. Thus, the quantitative results from MS-based methods are more reliable and accurate. These properties are summarized in Table 4. Clearly, immunoassays are low in cost and highly efficient. After all, for severe HF patients, time is life.
Improvement of BNP clinical testing
For improved testing, cross-reaction and lack of standardization should be resolved as soon as possible. Here, for the purpose of reducing negative effects on BNP measurement, some suggestions related to materials, methods and analytical devices have been discussed and examined.
According to the guidelines of ISO 17511, in a clinical testing laboratory, the calibrator, which is also referred to as standard material or reference material, is an essential material for obtaining a metrologically traceable measurement value . However, the development of a reference material is a complicated and rigorous scientific task. According to the data from the Joint Committee for Traceability in Laboratory Medicine, only 30 types of clinical proteins have been standardized to date . Technologies such as amino acid analytical methods based on isotope dilution MS and quantitative nuclear magnetic resonance methods are commonly adopted for impurity analysis and mass balance quantification. Once the purity of the candidate material is verified, it can be used for plasma BNP reference material preparation or quantification. Then, manufacturers would adopt this material for device calibration, and it would be applied for clinical measurement.
Currently, metrology institutes such as the Laboratory of the Government Chemist (LGC, UK) and the National Institute of Metrology (China) are focusing on this research. LGC has published works related to the development of the BNP reference quantitative MS method . Once a BNP 1-32 purity reference material is developed, it can provide a standard value for instrument calibration. Then no matter which immunoassay platform was selected, the measurement results could be traced to a reference value at any place and in any time, improving the measurement consistency.
Cleanup could improve the analytical sensitivity and maintain the molecular stability. A method for the rapid isolation of BNP from DPP-IV and amino- and carboxypeptidases is strongly needed for BNP 1-32 analysis. At present, a variety of commercial products are available for BNP purification. Hawkridge et al.  used a C8 SPE column and dual-antibody approach to purify BNP from plasma. They found that the highest percentage recovery was approximately 10%, and recovery improved as the incubation time was reduced and the primary antibody concentration was increased. Although no greater purification was achieved by any antibody, Lewis et al.  also found that the matrix effects were alleviated when using a C18 SPE to treat human plasma. Andrews et al.  developed an online analytical platform that includes cleanup, digestion, separation and MS detection. The results indicated that such an integrated online analytical approach is suitable for the measurement of low-abundance proteins.
In Tables 2 and 3, as the capture antibody could also bind to the metabolites of BNP 1-32 and proBNP, these methods exhibited a low specificity. However, the subsequent coupling to LC and MS devices allowed these targets to be conveniently separated and detected based on their polarity and relative molar mass. An immunoassay approach cannot recognize such NPs as easily as LC-MS. Therefore, the development of a BNP 1-32 enrichment approach could further increase the detection sensitivity and might decrease the cross-reactions in immunoassays.
To avoid undesirable cross-reactions, the development of novel antibodies might be an effective solution. Thus, the preparation of amino-terminal antibodies is attractive. Here, another type of antibody, termed an aptamer, will be introduced.
Aptamers are single-stranded DNA or RNA oligonucleotides generated from an in vitro selection process termed SELEX, which stands for systematic evolution of ligands by exponential enrichment (Figure 3). This approach, independently proposed by three groups in 1990 , , , aims to develop aptamers that bind to their target with high selectivity and sensitivity owing to their three-dimensional structure. The high specific affinity of an aptamer to its target is thought to resemble that of chemical antibodies, with dissociation constants ranging from the nanomolar to picomolar levels . Because of their high specific affinity, aptamers have broad applications that are undergoing rapid development in a variety of fields, such as disease diagnosis , , drug screening , bioseparation and bioanalysis , ,  and RNA computing .
Hence, the selection of aptamers that specifically recognize the amino-terminal sequence might reduce cross-reaction. Once BNP 1-32 and the aptamer form a complex, the steric hindrance might block the common epitope, preventing the recognition of proBNP by this aptamer. Because of a lack of serine-proline or serine-proline-lysine-methionine sequences, truncated BNPs, such as BNP 3-32 and BNP 5-32, would also not be recognized. Although this research is ongoing in the authors’ laboratory right now, previous inspiring results were reported by the Lewis group . In brief, Lewis et al.  raised and utilized a homemade polyclonal antibody that recognizes BNP 1-13 to decrease the cross-reaction with proBNP.
In addition to specificity and sensitivity, aptamers are also low-cost and easily modified molecules. Aptamers can be produced easily by polymerase chain reaction and can be conveniently modified with functional groups or linkers. Based on these characteristics, an aptamer-derived IVD reagent might be a promising tool for BNP 1-32 analysis without cross-reaction.
One limitation of clinical MS application is that biomarkers usually have low or ultralow abundance in biological samples . Although MS provides excellent sensitivity in select ion monitor mode or with an advanced mass analyzer, the cost is usually very high. Therefore, it is important to develop superchargers to increase the MS intensity , . Dimethyl sulfoxide (DMSO) is a supercharger well characterized by Hahne et al. . They found that a low percentage of DMSO in LC solvents led to a strong enhancement of the electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. For BNP quantification, Torma et al.  also observed that DMSO could enhance the intensity of +6 and +7 charge ions of intact BNP after postcolumn addition.
Because there are two cysteines in intact BNP, their modification needs to be considered. Cysteine alkylation could increase the ionization efficiency and improve MS quantitation . Shuford et al.  synthetized several types of hydrophobic alkylation reagents and used them for BNP alkylation. The isotope dilution MS results of BNP demonstrated that an adequate alkylation of BNP could lower the limit of detection and is especially suitable for the quantification of low-abundance proteins based on MS technology.
Ion mobility MS
Synthetic peptides or proteins usually contain a variety of similarly structured impurities. For instance, according to the results of the international Key Comparison Project (CCQM K115), 81 types of peptide-related impurities existed in the synthetic human C-peptide sample, including six isomers . Globally, pharmacopeia in different countries have also defined the threshold value of impurity for peptide drugs.
Before the invention of ion mobility MS (IM-MS), LC or CE was the common analytical approach for impurity separation. As a powerful complementary analytical approach to nuclear magnetic resonance and X-ray crystallography, IM-MS has more recently been widely applied in the field of structure characterization, with applications involving chemical warfare agents , supplements , , peptide measurement , , disease research , , drugs  and even protein complex conformation , . SYNAPT HDMS is the first commercialized IM-MS device in the world and has the potential to analyze isomer ions because a drift tube is located at the front of the TOF analyzer. Briefly, the ions in the drift tube undergo physical collision with nitrogen or argon, and then the isomers with different cross-sections will arrive at the analyzer at distinct times.
Because mobility separation can be achieved in milliseconds, IM-MS is highly suitable for the quality control of synthetic BNP 1-32. For instance, Bros et al.  investigated the disulfide bond mismatch situation when using SYNAPT G2-S HDMS to analyze synthetic hepcidin, which possesses four pairs of disulfide bonds in the native structure. The sequence could also be identified by the ion fragmentation after IM separation. A rapid and reliable structural characterization method is beneficial for accurate quantification.
In recent years, some advanced electrospray ion sources have been commercialized and have demonstrated a robust ability to handle complicated matrix samples. Ambient mass spectrometry (AMS)  is undoubtedly a breakthrough in the MS field, and the most representative AMS techniques are desorption electrospray ionization (DESI)  (Figure 4) and direct analysis in real time . Because of the absence of tedious sample preparation and time-consuming chromatographic separation, this technique provides great convenience for the rapid and straightforward analysis of target analytes in their native conditions , which is very attractive for the high-throughput screening and analysis of molecules with short half-lives, such as plasma BNP. Among all ion sources, spray-based ionization techniques, such as DESI and paper spray (PS), are the most popular techniques for blood or serum analysis in clinical chemistry.
Generally, in DESI, a fine spray of charged droplets bombards the surface of the sample, where the droplets pick up analyte molecules, ionize them in the gas phase and deliver them into the MS for further analysis. Wiseman et al.  used DESI to quantitatively determine xenobiotics in whole blood samples without sample preparation or chromatography. The collected whole blood was spotted onto specialty paper and dried to obtain dried blood spots. In addition to small molecules, DESI can ionize proteins as large as 150 kDa, such as superoxide dismutase, enolase, hemoglobin, cytochrome c, gramicidin D and immunoglobulin G . In this study, liquid sample DESI was used directly for the analysis of solutions; therefore, blood could be directly ionized without spotting and drying.
In PS, the sample is loaded on triangular paper substrates with a sharp point, and a stable spray is formed with the application of spray solvent and a high electric field on wet paper . The porous paper substrate plays three roles: sample collection, analyte separation and ionization. In contrast to DESI, the paper introduces chromatographic separation to decrease matrix effects, so PS showed better performance in complex mixture analysis. Surface modification of the paper can greatly enhance its capability as a separation medium, as illustrated by silica-coated paper , zirconia-coated paper  and silver nanoparticle-coated paper .
Ambient ion sources are highly convenient for sample analysis, but the lower ionization efficiency severely restricts their further application in ultratrace analyte determination. Obviously, the detection of BNP in blood is a challenging task, and there are no reports to date. Therefore, sample preparation strategies  have been developed to improve the practical utility of techniques for the detection of ultratrace analytes, including SPE , solid-phase microextraction  and immunoaffinity. Chen and Badu-Tawiah  built a PS-based immunoassay platform for large molecule detection, including malaria antigen and cancer antigen. High selectivity was achieved by antigen and antibody interaction, and high ionization efficiency was achieved by the use of cleavable ionic probes as a mass reporter. The reliability of results is strongly dependent on the internal standard; therefore, the development of a BNP reference material is essential for such advanced DESI analysis.
MS provides excellent selectivity, precision and accuracy in measurement, and the development of MS instrumentation and informatics tools has set the stage for protein analysis to emerge in the clinical laboratory , . Because many NPs coexist in the circulation of HF patients, additional separation was commonly needed before MS profiling. Fortunately, in recent years, some technologies, such as IM-MS and DESI ionization, have provided direct analytical ability, which would reduce the complexity of clinical measurement.
In essence, the structural diversity of circulating NPs leads to overestimated immunoassay results. In addition, the lack of standardization capacity is the main reason for the poor harmonization of clinical measurement results. Both situations could introduce error into the measurement results; however, the approaches to resolution are distinct. The development of amino-terminal-specific recognition or detection ligands is an effective method for reducing cross-reactions. Meanwhile, research on SI-traceable reference materials or reference measurement procedures will increase measurement consistency. In practice, however, the mentioned resolving approaches in this review might be helpful in the standardization of a specific BNP or an unprocessed proBNP measurement, but not suitable for the standardization of multiple BNP and proBNP measurement in clinical. Clinically relevant BNP is represented by a mixture of BNP-related peptides, and the analysis of such very heterogeneous analyte is a challenge for clinical researchers; therefore, although analytical technology has been improved rapidly in the current, there is still a long way for the standardization of measurement of BNP-related peptides.
The authors acknowledge financial support from the National Nature Science Foundation of China (21705144) and the National Key R&D Program of China (2017YFF0205401).
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About the article
Published Online: 2018-11-24
Published in Print: 2019-06-26
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This work was supported by the National Nature Science Foundation of China (21705144) and the National Key R&D Program of China (2017YFF0205401).
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.