A review of intact glycopeptide enrichment and glycan separation through hydrophilic interaction liquid chromatography stationary phase materials.

Protein glycosylation, one of the most important biologically relevant post-translational modifications for biomarker discovery, faces analytical challenges due to heterogeneous glycosite, diverse glycans, and mass spectrometry limitations. Glycopeptide enrichment by removing abundant hydrophobic peptides helps overcome some of these obstacles. Hydrophilic interaction liquid chromatography (HILIC), known for its selectivity, glycan separations, intact glycopeptide enrichment, and compatibility with mass spectrometry, has seen recent advancements in stationary phases like Amide-80, glycoHILIC, amino acids or peptides for improved HILIC-based glycopeptide analysis. Utilization of these materials can improve glycopeptide enrichment through solid-phase extraction and separation via high-performance liquid chromatography. Additionally, using glycopeptides themselves to modify HILIC stationary phases holds promise for improving selectivity and sensitivity in glycosylation analysis. Additionally, HILIC has capability to assess the information about glycosites and structural information of glycans. This review summarizes recent breakthroughs in HILIC stationary materials, highlighting their impact on glycopeptide analysis. Ongoing research on advanced materials continues to refine HILIC's performance, solidifying its value as a tool for exploring protein glycosylation.

[Extraction, isolation, structural characterization and pharmacological effects of glycopeptides from traditional Chinese medicine: current status and prospects].

Glycopeptides of traditional Chinese medicine(GTCM), as a type of natural products with important biological activities, have received increasing attention in recent years. These substances have a variety of pharmacological effects, including anti-tumor, immunomodulatory, neuroprotective, and anti-inflammatory effects. The extraction and separation processes directly affect the yield and purity of GTCM, and structural characterization is essential for probing into the properties and pharmacological mechanisms of glycopeptides. This article reviews the research progress and prospects the research directions in the extraction, separation, structural identification, and pharmacological effects of GTCM. Despite the progress in the research on GTCM, challenges such as low extraction efficiency, long separation cycles, difficult structural characterization, and complex mechanisms still exist. To address these issues, efforts should be made to optimize the extraction methods, explore new separation technologies, and develop efficient structural characte-rization methods. Additionally, the future work should decipher the pharmacological mechanisms of GTCM, which will provide a scientific basis for the drug development and clinical applications of GTCM.

LC-MS/MS of isomeric N-and O-glycopeptides on mesoporous graphitized carbon column.

BACKGROUND: The study of glycopeptides is associated with challenges regarding the microheterogeneity of different isomeric glycans occupying the same glycosylation sites in glycoproteins. It is immensely valuable to perform both qualitative and quantitative site-specific glycosylation analysis of glycopeptide isomers due to their link to several diseases. Achieving isomeric separation of glycopeptides is particularly challenging due to the low abundance of glycopeptides as well as inefficient ionization. Although some methods have demonstrated the isomeric separation of glycopeptides, a more efficient nanoflow-based stationary phase is needed for the isomeric separation of both N- and O-glycopeptides. RESULTS: In this study, the separation of N- and O-glycopeptide isomers at 75 °C was achieved with an in-house packed 1 cm long mesoporous graphitized carbon (MGC) column. Different gradient compositions of the optimized mobile phase for separating permethylated glycans on MGC column were tested, and we observed efficient separation of N- and O-glycopeptide isomers at a gradient elution time of 120 min. After achieving the isomeric separation of sialylated glycopeptides from model glycoproteins derived from bovine fetuin, the separation of isomeric glycopeptides derived from asialofetuin, α-1 glycoprotein and human blood serum were also demonstrated. Furthermore, the developed method for the separation of isomeric N- and O-glycopeptide on MGC column showed high reproducibility over three months. We observed an average retention time shift of 1 min and consistent resolution of separated peaks throughout three months. SIGNIFICANCE AND NOVELTY: MGC column can serve as an efficient tool for obtaining the isomeric separation of N- and O-glycopeptide from complex biological samples in future studies. This will enable a more profound understanding of the roles played by isomeric N- and O-glycopeptide in important biological processes and their correlations to various disease progressions.

A Fully Automated Online Enrichment and Separation System for Highly Reproducible and In-Depth Analysis of Intact Glycopeptide.

A fully automated online enrichment and separation system for intact glycopeptides, named AutoGP, was developed in this study by integrating three different columns in a nano-LC system. Specifically, the peptide mixture from the enzymatic digestion of a complex biological sample was first loaded on a hydrophilic interaction chromatography (HILIC) column. The nonglycopeptides in the sample were washed off the column, and the glycopeptides retained by the HILIC column were eluted to a C18 trap column to achieve an automated glycopeptide enrichment. The enriched glycopeptides were further eluted to a C18 column for separation, and the separated glycopeptides were eventually analyzed by using an orbitrap mass spectrometer (MS). The optimal operating conditions for AutoGP were systemically studied, and the performance of the fully optimized AutoGP was compared with a conventional manual system used for glycopeptide analysis. The experimental evaluation shows that the total number of glycopeptides identified is at least 1.5-fold higher, and the median coefficient of variation for the analyses is at least 50% lower by using AutoGP, as compared to the results acquired by using the manual system. In addition, AutoGP can perform effective analysis even with a 1-µg sample amount, while a 10-µg sample at least will be needed by the manual system, implying an order of magnitude better sensitivity of AutoGP. All the experimental results have consistently proven that AutoGP can be used for much better characterization of intact glycopeptides.

Genome mining methods to discover bioactive natural products.

Covering: 2016 to 2021With genetic information available for hundreds of thousands of organisms in publicly accessible databases, scientists have an unprecedented opportunity to meticulously survey the diversity and inner workings of life. The natural product research community has harnessed this breadth of sequence information to mine microbes, plants, and animals for biosynthetic enzymes capable of producing bioactive compounds. Several orthogonal genome mining strategies have been developed in recent years to target specific chemical features or biological properties of bioactive molecules using biosynthetic, resistance, or transporter proteins. These "biosynthetic hooks" allow researchers to query for biosynthetic gene clusters with a high probability of encoding previously undiscovered, bioactive compounds. This review highlights recent case studies that feature orthogonal approaches that exploit genomic information to specifically discover bioactive natural products and their gene clusters.

Fabrication of magnetic dual-hydrophilic metal organic framework for highly efficient glycopeptide enrichment.

Highly selective glycopeptide enrichment is important before mass spectrometry analysis because of the ultra-low abundance of glycopeptides in the peptide mixtures. Herein, a UiO-66-NH2-based magnetic composite was prepared and used for the hydrophilic enrichment of glycopeptides. The composite was modified with phytic acid (PA) molecules by partially replacing 2-aminoterephthalic acid ligands in UiO-66-NH2, with electrostatic interactions also promoting this modification process. Based on the hydrophilicity of both the PA molecules and the UiO-66-NH2 skeleton, the resulting material, denoted as MUiO-66-NH2/PA, showed excellent dual hydrophilicity towards glycopeptide enrichment. Compared with pure UiO-66-NH2, the specific surface area and hydrophilicity of the prepared material were increased, and MUiO-66-NH2/PA exhibited good magnetic responsiveness to facilitate a convenient enrichment procedure. HRP and IgG were used as standard proteins to evaluate the glycopeptide enrichment properties, with 21 and 34 glycopeptides enriched from their tryptic digests. Furthermore, MUiO-66-NH2/PA showed outstanding sensitivity (1 fmol/µL) and selectivity (HRP/BSA = 1:1000), and achieved remarkable glycopeptide enrichment performance for practical human serum samples. Notably, MUiO-66-NH2/PA showed perfect reusability and stability, achieving enrichment performance after five cycles similar to that of the first use. This material can be used for glycopeptide enrichment to obtain further glycosylation information, providing the possibility for cancer treatment.

Glutathione-functionalized two-dimensional cobalt sulfide nanosheets for rapid and highly efficient enrichment of N-glycopeptides.

Protein glycosylation plays pivotal role in a variety of biological processes and has association with many diseases. The highly efficient glycopeptide enrichment is essential for the mass spectrometry-based glycoproteome research to reduce interference from non-glycopeptides. In this study, novel glutathione-functionalized two-dimensional cobalt sulfide nanosheets (Co-S@Au-GSH) were synthesized for rapid and highly effective enrichment of glycopeptides. By using this nanomaterial, 34 and 21 N-glycopeptides were effectively captured from human serum immunoglobulin G (IgG) and horseradish peroxidase (HRP) digests, respectively. In addition, the Co-S@Au-GSH showed remarkable performance in N-glycopeptide extraction with high selectivity (HRP: BSA = 1:500), low limit of detection (0.5 fmol/µL), high binding capacity (150 mg/g), good reusability, and great robustness. Moreover, it was successfully applied in complex serum samples, demonstrating its excellent enrichment performance. These results indicated that this nanomaterial has great potential in complicated practice samples in glycoproteome determination.

Simultaneous enrichment and separation of neutral and sialyl glycopeptides of SARS-CoV-2 spike protein enabled by dual-functionalized Ti-IMAC material.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein's glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry.

Facile synthesis of bifunctional polymer monolithic column for tunable and specific capture of glycoproteins and phosphoproteins.

Efficiently tunable capture of the glycosylated/phosphorylated proteins is critical to meet the need of in-depth glycoproteome and phosphoproteome studies. Reported here is a new bifunctional polymer monolithic column by introducing benzeneboronic acid and phosphonic acid onto monolithic column (denoted as poly (EDMA-co-VPBA-co-VPA) monolith) for tunable and specific enrichment of glycoproteins and phosphoproteins via switching different mobile phases. Based on boronate affinity and immobilized metal affinity, the as-prepared poly (EDMA-co-VPBA-co-VPA) monolith exhibited superior performance in selective separation of small molecules and biomacromolecules containing cis-diol/phosphate groups or not. And the frontal chromatography analysis showed that the binding capacity of the poly (EDMA-co-VPBA-co-VPA) monolith towards horseradish peroxidase (HRP, glycoprotein) or ß-casein (phosphoprotein) is four-fold higher than that of bovine serum albumin (BSA, non-glycosylated/phosphorylated protein). Furthermore, combined with mass spectrometry identification, the successful application in specific enrichment of glycopeptides/phosphopeptides from tryptic digests of HRP/ß-casein and direct capture of low abundant endogenous phosphopeptides from human serum proved great practicability in complex samples. This study provides a novel insight for fabricating the monolithic columns with multifunctionalization to facilitate further post-translational modification (PTM)-proteomics development.

Comparison of human IgG glycopeptides separation using mixed-mode hydrophilic interaction/ion-exchange liquid chromatography and reversed-phase mode.

Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)- with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica- with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.

Mycobacterium abscessus biofilms have viscoelastic properties which may contribute to their recalcitrance in chronic pulmonary infections.

Mycobacterium abscessus is emerging as a cause of recalcitrant chronic pulmonary infections, particularly in people with cystic fibrosis (CF). Biofilm formation has been implicated in the pathology of this organism, however the role of biofilm formation in infection is unclear. Two colony-variants of M. abscessus are routinely isolated from CF samples, smooth (MaSm) and rough (MaRg). These two variants display distinct colony morphologies due to the presence (MaSm) or absence (MaRg) of cell wall glycopeptidolipids (GPLs). We hypothesized that MaSm and MaRg variant biofilms might have different mechanical properties. To test this hypothesis, we performed uniaxial mechanical indentation, and shear rheometry on MaSm and MaRg colony-biofilms. We identified that MaRg biofilms were significantly stiffer than MaSm under a normal force, while MaSm biofilms were more pliant compared to MaRg, under both normal and shear forces. Furthermore, using theoretical indices of mucociliary and cough clearance, we identified that M. abscessus biofilms may be more resistant to mechanical forms of clearance from the lung, compared to another common pulmonary pathogen, Pseudomonas aeruginosa. Thus, the mechanical properties of M. abscessus biofilms may contribute to the persistent nature of pulmonary infections caused by this organism.

Hydrophilic carrageenan functionalized magnetic carbon-based framework linked by silane coupling agent for the enrichment of N-glycopeptides from human saliva.

In this work, a magnetic graphene material coated with mesoporous silica was selected as the substrate, 3-glycidoxypropyltrimethoxysilane and polyethyleneimine were sequentially bonded through chemical reactions, and then carrageenan was successfully introduced by electrostatic interaction; finally, hydrophilic nanocomposite material was prepared. Due to the large number of hydrophilic groups, and polyethyleneimine was connected by means of chemical bonds, this material exhibits good hydrophilicity and stability for glycopeptide enrichment. In the actual enrichment process, nanomaterial exhibits high selectivity (1:500), high sensitivity (2 fmol), and good repeatability (five cycles). In addition, the synthesized material also shows a good enrichment effect in the face of actual complex biological samples, which captured 40 N-glycopeptides from human saliva, indicating the application potential for enrichment of N-glycopeptides.

Hollow MnFe2O4@C@APBA Nanospheres with Size Exclusion and pH Response for Efficient Enrichment of Endogenous Glycopeptides.

Enrichment and detection of glycopeptides are an important clinical measure for the diagnosis of complex diseases. Enrichment materials play a key role in this process; they must have an effective sample-screening ability to eliminate the interference of nonglycopeptides. In this work, novel hollow MnFe2O4@C@APBA nanospheres (HMCAs) with magnetic and pH responsiveness were prepared for glycopeptide enrichment. The as-prepared composites have a suitable hollow structure and large specific surface area, and the boron hydroxyl group in their cavities can fix or disconnect the hydrophilic groups of the glycopeptides at different pH, so the glycopeptides can be adsorbed or desorbed in a controllable way. Enrichment results showed that the HMCAs exhibited an excellent enrichment performance: ultralow limit of detection (approximately 0.5 fmol µL-1), perfect size-exclusion effect (HRP/BSA, 1:800, w/w), favorable universality (HRP, IgG, and RNase B), and high binding capacity (150 mg/g). In order to verify the application of materials in practice, the HMCAs were used for the analysis of complex samples and it was found that 474 glycopeptides were identified from 210 glycoproteins in three replicate analyses of 2 µL of human serum. The results showed that the HMCAs could be used as a promising enrichment material for glycopeptide characterization in MS-based glycoproteomics and related fields.

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides.

Changes in the glycome of human proteins and cells are associated with the progression of multiple diseases such as Alzheimer's, diabetes mellitus, many types of cancer, and those caused by viruses. Consequently, several studies have shown essential modifications to the isomeric glycan moieties for diseases in different stages. However, the elucidation of extensive isomeric glycan profiles remains challenging because of the lack of analytical techniques with sufficient resolution power to separate all glycan and glycopeptide iso-forms. Therefore, the development of sensitive and accurate approaches for the characterization of all the isomeric forms of glycans and glycopeptides is essential to tracking the progression of pathology in glycoprotein-related diseases. This review describes the isomeric separation achievements reported in glycomics and glycoproteomics in the last decade. It focuses on the mass spectrometry-based analytical strategies, stationary phases, and derivatization techniques that have been developed to enhance the separation mechanisms in liquid chromatography systems and the detection capabilities of mass spectrometry systems.

Comparison of Different HILIC Stationary Phases in the Separation of Hemopexin and Immunoglobulin G Glycopeptides and Their Isomers.

Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.

Gold nanoparticle-glutathione-functionalized porous graphene oxide-based hydrophilic beads for the selective enrichment of N-linked glycopeptides.

A three-dimensional structured porous graphene oxide-polyethylenimine bead (pGP) is synthesized for immobilizing gold nanoparticles and modifying glutathione molecules (denoted as pGP/AuG). The pGP/AuG has open pore structure, honeycomb-like channels, and excellent hydrophilicity. By taking advantages of the porous structure, abundant binding sites, and multivalent interactions between glycopeptides and both glutathione molecules and free amino groups, the pGP/AuG is adopted to the selective enrichment of N-linked glycopeptides with low limit of detection (2 fmol), high enrichment selectivity (1:500), binding capacity (333.3 mg/g), recovery yield (91.3 ± 2.1%), and repeatability (< 6.0% RSD) using matrix-assisted laser desorption/ionization time of flight mass spectrometry detection method. Furthermore, the practical applicability of pGP/AuG is evaluated, in which 209 N-glycosylated peptides corresponding to 128 N-glycosylated proteins are identified from 1 µL human serum in three independent analysis procedures, suggesting the great potential for application in glycoproteome fields.Graphical abstract Schematic presentation of preparation for porous graphene oxide-based hydrophilic beads (pGP/AuG) with honeycomb-like microstructure. The pGP/AuG was successfully used for enriching and identifying glycopeptides from actual biological sample.

Glycan-specific precipitation of glycopeptides in high organic content sample solvents used in HILIC.

The composition of a sample solvent has a crucial impact on separations in hydrophilic interaction liquid chromatography (HILIC). In this short communication, we studied the effect of an organic modifier in the sample solvent on the solubility of different tryptic glycopeptides of hemopexin and haptoglobin proteins. The results showed that the solubility of glycopeptides in solvents with a high acetonitrile content depends on the type of attached N-glycan. We observed lower solubility in larger glycans attached to the same peptide backbone, and we demonstrated that glycopeptides containing sialic acids precipitate more readily than those without sialic acid. Therefore, the sample solvent composition in HILIC must be carefully optimized for accurate quantitative data collection and for adequate separation.

Preparation of a biomimetic ionic liquids hybrid polyphosphorylcholine monolithic column for the high efficient capillary microextraction of glycopeptide antibiotics.

An ionic liquid hybrid zwitterionic polymer capillary microextraction (CME) column was prepared for the biomimetic enrichment of glycopeptides by one-step copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 1-butyl-3-vinylimidazolium bromide, in the presence of crosslinker trimethylolpropane trimethacrylate (TMA). The resultant monolith was characterized by scanning electron microscopy (SEM), fourier transform infrared (FT-IR) spectroscopy and pore size distribution measurement. Due to the incorporation of zwitterionic MPC owning a unique biomimic structure (i.e. hydrophilic cation/anion and hydrophobic long-alkyl chain), the monolithic column has large pore size and good biocompatibility, exhibiting high extraction efficiency, permeability and fast mass transfer to targets. Besides, the use of ionic liquids (ILs) as co-monomer in the polymerization endows the monolith with enhanced mechanical stability, uniformity and multiple interactions. The prepared column was successfully applied in CME coupled to capillary electrochromatography (CEC) for the efficient enrichment and separation of glycopeptide antibiotics in foodstuff. The method demonstrated a wide linear range (50.0-18000.0 µg L-1), low detection limits (5.0-10.0 µg L-1, S/N = 3) and satisfied recoveries (76.0-109.7%). This work shows the advantage of fine-tuning biomimetic monoliths in application-specific CME-CEC.

Cacaoidin, First Member of the New Lanthidin RiPP Family.

Lantibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d-amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N-dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram-positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.

Recovery of value-added glycopeptides from edible bird's nest (EBN) co-products: enzymatic hydrolysis, physicochemical characteristics and bioactivity.

BACKGROUND: Processing of edible bird's nest (EBN) requires extensive washing to remove impurities and produces huge amounts of EBN co-products, which contain mainly feathers with glycoproteins attached, which are usually discarded. This study was conducted to recover the valuable EBN glycoproteins from the waste material. Enzymatic hydrolysis was applied to recover EBN glycopeptides from EBN co-products (EBNcoP ) and processed cleaned EBN (EBNclean ) was used as control, which were then freeze-dried into EBN hydrolysates (EBNhcoP and EBNhclean , respectively). RESULTS: The recovery yield for EBNhclean and EBNhcoP were 89.09 ± 0.01% and 47.64 ± 0.26%, respectively, indicating nearly 50% of glycopeptide can be recovered from the waste material. Meanwhile, N-acetylneuraminic acid, a major acid sugar in EBN glycoproteins, of EBNhcoP increased by 229% from 58.6 ± 3.9 to 192.9 ± 3.1 g kg-1 , indicating the enzymatic hydrolysis removed impurities and thus enhanced the N-acetylneuraminic acid content. Total soluble protein was more than 330 g kg-1 for all the samples. Colour parameter showed that hydrolysate samples have greater L* (lightness) values. Chroma result indicates the intensity of all the samples were low (< 11). Fourier-transform infrared (FTIR) spectrum displayed that the EBNhcoP exhibited similar functional groups with EBNhclean , indicating that the EBNcoP has similar functionality as EBNclean . Significantly higher (P ≤ 0.05) 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) activities were reported in EBNhcoP after the enzymatic reaction. CONCLUSION: EBNhcoP were successfully recovered from low value EBNcoP with enhanced antioxidant activities. The findings of this work are beneficial for the EBN industry to reduce wastage and enhance economic values of EBN co-products, both economically and nutritionally. © 2020 Society of Chemical Industry.