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

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.