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Dynamic degradation and glycoform heterogeneity of NT-proBNP in serum: Implications for biomarker quantification.

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Τίτλος: Dynamic degradation and glycoform heterogeneity of NT-proBNP in serum: Implications for biomarker quantification.
Συγγραφείς: Wu J; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China., Ma X; Beijing Rongzhi Youpu Biotechnology Co Ltd, Daxing, Beijing, China., Dong Y; Beijing Rongzhi Youpu Biotechnology Co Ltd, Daxing, Beijing, China., Ma Y; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China., Zhang X; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China., Liu Z; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China., Yuan H; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China. Electronic address: yuanyuan@mail.ccmu.edu.cn.
Πηγή: International journal of biological macromolecules [Int J Biol Macromol] 2026 Jun; Vol. 366, pp. 152515. Date of Electronic Publication: 2026 May 12.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Elsevier Country of Publication: Netherlands NLM ID: 7909578 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1879-0003 (Electronic) Linking ISSN: 01418130 NLM ISO Abbreviation: Int J Biol Macromol Subsets: MEDLINE
Imprint Name(s): Publication: Amsterdam : Elsevier
Original Publication: Guildford, Eng., IPC Science and Technology Press.
Ιατρικοί όροι (MeSH): Natriuretic Peptide, Brain*/blood , Natriuretic Peptide, Brain*/chemistry , Natriuretic Peptide, Brain*/metabolism , Peptide Fragments*/blood , Peptide Fragments*/chemistry , Peptide Fragments*/metabolism, Biomarkers/blood ; Humans ; Glycosylation ; Proteolysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Περίληψη: N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a widely used biomarker for heart failure, yet its circulating molecular heterogeneity and susceptibility to ex vivo proteolysis complicate accurate and traceable quantification. In this study, we systematically investigated the degradation behavior of NT-proBNP in human serum and identified kinetically stable endogenous glycoforms using immunoaffinity enrichment coupled with MALDI-TOF mass spectrometry. Non-glycosylated recombinant NT-proBNP spiked into serum underwent rapid proteolytic processing, dominated by stepwise C-terminal truncation, with extensive fragmentation observed within hours even at 4 °C. Protease inhibition experiments performed under accelerated incubation conditions (37 °C, up to 48 h) confirmed that these changes were enzyme-driven. Against this dynamic background, two NT-proBNP-related peaks at m/z 9084 and 9517 remained qualitatively detectable throughout the entire incubation series, whereas labile non-glycosylated forms showed rapid attenuation. These peaks were reproducibly detected in the small NT-proBNP-positive sample set and were not detected in the small NT-proBNP-negative comparison set, supporting preliminary analytical specificity in this pilot dataset. Mass-balance modeling suggested that both species may correspond to proteolytically processed NT-proBNP backbones carrying clustered mucin-type O-glycosylation involving GalNAc, hexose, and sialic acid residues; these assignments should be regarded as putative compositions requiring orthogonal structural validation. Together, these results suggest that O-glycosylation may contribute to the qualitative persistence of specific NT-proBNP-related species in serum. The m/z 9084 and 9517 peaks should therefore be regarded as candidate, analytically persistent signals detected under the present 15F11-based immunoenrichment and MALDI-TOF MS workflow. Their potential use as robust analytical targets or anchors remains a hypothesis that will require orthogonal structural confirmation, quantitative validation, and evaluation in larger cohorts.
(Copyright © 2026 Elsevier B.V. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Biomarker stability; Mass spectrometry; O-linked glycosylation; Proteolytic degradation; Serum kinetics
Substance Nomenclature: 114471-18-0 (Natriuretic Peptide, Brain)
0 (Biomarkers)
0 (Peptide Fragments)
0 (pro-brain natriuretic peptide (1-76))
Entry Date(s): Date Created: 20260513 Date Completed: 20260716 Latest Revision: 20260716
Update Code: 20260717
DOI: 10.1016/j.ijbiomac.2026.152515
PMID: 42128113
Βάση Δεδομένων: MEDLINE
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  – Url: https://www.doi.org/10.1016/j.ijbiomac.2026.152515?
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  Data: Dynamic degradation and glycoform heterogeneity of NT-proBNP in serum: Implications for biomarker quantification.
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  Data: <searchLink fieldCode="AU" term="%22Wu+J%22">Wu J</searchLink>; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China.<br /><searchLink fieldCode="AU" term="%22Ma+X%22">Ma X</searchLink>; Beijing Rongzhi Youpu Biotechnology Co Ltd, Daxing, Beijing, China.<br /><searchLink fieldCode="AU" term="%22Dong+Y%22">Dong Y</searchLink>; Beijing Rongzhi Youpu Biotechnology Co Ltd, Daxing, Beijing, China.<br /><searchLink fieldCode="AU" term="%22Ma+Y%22">Ma Y</searchLink>; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China.<br /><searchLink fieldCode="AU" term="%22Zhang+X%22">Zhang X</searchLink>; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China.<br /><searchLink fieldCode="AU" term="%22Liu+Z%22">Liu Z</searchLink>; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China.<br /><searchLink fieldCode="AU" term="%22Yuan+H%22">Yuan H</searchLink>; Department of Clinical Laboratory, Beijing Anzhen Hospital, Capital Medical University, Chaoyang, Beijing, China. Electronic address: yuanyuan@mail.ccmu.edu.cn.
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  Data: <searchLink fieldCode="JN" term="%227909578%22">International journal of biological macromolecules</searchLink> [Int J Biol Macromol] 2026 Jun; Vol. 366, pp. 152515. <i>Date of Electronic Publication: </i>2026 May 12.
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  Data: English
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22Elsevier%22">Elsevier </searchLink><i>Country of Publication: </i>Netherlands <i>NLM ID: </i>7909578 <i>Publication Model: </i>Print-Electronic <i>Cited Medium: </i>Internet <i>ISSN: </i>1879-0003 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2201418130%22">01418130 </searchLink><i>NLM ISO Abbreviation: </i>Int J Biol Macromol <i>Subsets: </i>MEDLINE
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  Data: <i>Publication</i>: Amsterdam : Elsevier<br /><i>Original Publication</i>: Guildford, Eng., IPC Science and Technology Press.
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  Data: <searchLink fieldCode="MM" term="%22Natriuretic+Peptide%2C+Brain%22">Natriuretic Peptide, Brain*</searchLink>/<searchLink fieldCode="MM" term="%22Natriuretic+Peptide%2C+Brain+blood%22">blood</searchLink> <br /><searchLink fieldCode="MM" term="%22Natriuretic+Peptide%2C+Brain%22">Natriuretic Peptide, Brain*</searchLink>/<searchLink fieldCode="MM" term="%22Natriuretic+Peptide%2C+Brain+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22Natriuretic+Peptide%2C+Brain%22">Natriuretic Peptide, Brain*</searchLink>/<searchLink fieldCode="MM" term="%22Natriuretic+Peptide%2C+Brain+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Peptide+Fragments%22">Peptide Fragments*</searchLink>/<searchLink fieldCode="MM" term="%22Peptide+Fragments+blood%22">blood</searchLink> <br /><searchLink fieldCode="MM" term="%22Peptide+Fragments%22">Peptide Fragments*</searchLink>/<searchLink fieldCode="MM" term="%22Peptide+Fragments+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22Peptide+Fragments%22">Peptide Fragments*</searchLink>/<searchLink fieldCode="MM" term="%22Peptide+Fragments+metabolism%22">metabolism</searchLink><br /><searchLink fieldCode="MH" term="%22Biomarkers%22">Biomarkers</searchLink>/<searchLink fieldCode="MH" term="%22Biomarkers+blood%22">blood</searchLink> ; <searchLink fieldCode="MH" term="%22Humans%22">Humans</searchLink> ; <searchLink fieldCode="MH" term="%22Glycosylation%22">Glycosylation</searchLink> ; <searchLink fieldCode="MH" term="%22Proteolysis%22">Proteolysis</searchLink> ; <searchLink fieldCode="MH" term="%22Spectrometry%2C+Mass%2C+Matrix-Assisted+Laser+Desorption-Ionization%22">Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</searchLink>
– Name: Abstract
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  Data: N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a widely used biomarker for heart failure, yet its circulating molecular heterogeneity and susceptibility to ex vivo proteolysis complicate accurate and traceable quantification. In this study, we systematically investigated the degradation behavior of NT-proBNP in human serum and identified kinetically stable endogenous glycoforms using immunoaffinity enrichment coupled with MALDI-TOF mass spectrometry. Non-glycosylated recombinant NT-proBNP spiked into serum underwent rapid proteolytic processing, dominated by stepwise C-terminal truncation, with extensive fragmentation observed within hours even at 4 °C. Protease inhibition experiments performed under accelerated incubation conditions (37 °C, up to 48 h) confirmed that these changes were enzyme-driven. Against this dynamic background, two NT-proBNP-related peaks at m/z 9084 and 9517 remained qualitatively detectable throughout the entire incubation series, whereas labile non-glycosylated forms showed rapid attenuation. These peaks were reproducibly detected in the small NT-proBNP-positive sample set and were not detected in the small NT-proBNP-negative comparison set, supporting preliminary analytical specificity in this pilot dataset. Mass-balance modeling suggested that both species may correspond to proteolytically processed NT-proBNP backbones carrying clustered mucin-type O-glycosylation involving GalNAc, hexose, and sialic acid residues; these assignments should be regarded as putative compositions requiring orthogonal structural validation. Together, these results suggest that O-glycosylation may contribute to the qualitative persistence of specific NT-proBNP-related species in serum. The m/z 9084 and 9517 peaks should therefore be regarded as candidate, analytically persistent signals detected under the present 15F11-based immunoenrichment and MALDI-TOF MS workflow. Their potential use as robust analytical targets or anchors remains a hypothesis that will require orthogonal structural confirmation, quantitative validation, and evaluation in larger cohorts.<br /> (Copyright © 2026 Elsevier B.V. All rights reserved.)
– Name: Abstract
  Label: Competing Interests
  Group: Ab
  Data: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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  Data: <i>Keywords: </i>Biomarker stability; Mass spectrometry; O-linked glycosylation; Proteolytic degradation; Serum kinetics
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  Data: 114471-18-0 (Natriuretic Peptide, Brain)<br />0 (Biomarkers)<br />0 (Peptide Fragments)<br />0 (pro-brain natriuretic peptide (1-76))
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  Data: <i>Date Created: </i>20260513 <i>Date Completed: </i>20260716 <i>Latest Revision: </i>20260716
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  Data: 42128113
PLink https://search.ebscohost.com/login.aspx?direct=true&site=eds-live&db=cmedm&AN=42128113
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        Value: 10.1016/j.ijbiomac.2026.152515
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        Text: English
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      – SubjectFull: Humans
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      – SubjectFull: Glycosylation
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      – TitleFull: Dynamic degradation and glycoform heterogeneity of NT-proBNP in serum: Implications for biomarker quantification.
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              Text: 2026 Jun
              Type: published
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