Glycoproteomics: Charting new territory in mass spectrometry and glycobiology.

Glycosylation is an incredibly common and diverse post-translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision-based fragmentation, thus necessitating electron-based fragmentation for site-localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.

Ultrasound modification on milk fat globule membrane and soy lecithin to improve the physicochemical properties, microstructure and stability of mimicking human milk fat emulsions.

Starting from the consideration of the structure of human milk fat globule (MFG), this study aimed to investigate the effects of ultrasonic treatment on milk fat globule membrane (MFGM) and soy lecithin (SL) complexes and their role in mimicking human MFG emulsions. Ultrasonic power significantly affected the structure of the MFGM-SL complex, further promoting the unfolding of the molecular structure of the protein, and then increased solubility and surface hydrophobicity. Furthermore, the microstructure of mimicking MFG emulsions without sonication was unevenly distributed, and the average droplet diameter was large. After ultrasonic treatment, the droplets of the emulsion were more uniformly dispersed, the particle size was smaller, and the emulsification properties and stability were improved to varying degrees. Especially when the ultrasonic power was 300 W, the mimicking MFG emulsion had the highest encapsulation rate and emulsion activity index and emulsion stability index were increased by 60.88 % and 117.74 %, respectively. From the microstructure, it was observed that the spherical droplets of the mimicking MFG emulsion after appropriate ultrasonic treatment remain well separated without obvious flocculation. This study can provide a reference for the screening of milk fat globules mimicking membrane materials and the further utilization and development of ultrasound in infant formula.

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Stretchable Photonic Crystal-Assisted Glycoprotein Identification for Ovarian Cancer Diagnosis.

Photonic crystals with specific wavelengths can realize surface-enhanced excitation and emission intensities of fluorophores and enhance the fluorescence signals of fluorescent molecules. Herein, stretchable photonic crystals with good mechanochromic properties provide continuously adjustable forbidden wavelengths by stretching to change the lattice spacing, with reflectance peaks blue-shifted up to 110 nm to match indicators of different wavelengths and produce differentiated optical enhancement effects. Glycoproteins are significantly identified as clinical markers. However, the wide participation of glycoproteins in various life processes poses enormous complexity and critical challenges for rapid, facile, high-throughput, and accurate clinical analysis or health assessment. In this work, we proposed a stretchable photonic crystal-assisted glycoprotein identification approach for early ovarian cancer diagnosis. Stretchable photonic crystals can provide rich optical information to efficiently identify glycoproteins in complex matrices. A double-indicator fluorescence sensor was designed to respond to the protein trunk and oligosaccharide segment of glycoproteins separately for improved recognition accuracy. Seven typical glycoproteins could be discriminated from proteins, saccharides, or mixture interferents. Clinical ovarian cancer samples for early, intermediate, and advanced ovarian cancer and healthy subjects were verified with 100% accuracy. This strategy of stretchable photonic crystal-assisted glycoprotein identification provides an effective method for accurate, rapid ovarian cancer diagnosis and timely clinical treatment.

An XIC-Centric Strategy for Improved Identification and Quantification in Proteomic Data Analyses.

Reproducibility is a "proteomic dream" yet to be fully realized. A typical data analysis workflow utilizing extracted ion chromatograms (XICs) often treats the information path from identification to quantification as a one-way street. Here, we propose an XIC-centric approach in which the data flow is bidirectional: identifications are used to derive XICs whose information is in turn applied to validate the identifications. In this study, we employed liquid chromatography-mass spectrometry data from glycoprotein and human hair samples to illustrate the XIC-centric concept. At the core of this approach was XIC-based monoisotope repicking. Taking advantage of the intensity information for all detected isotopes across the whole range of an XIC peak significantly improved the accuracy and uncovered misidentifications originating from monoisotope assignment mistakes. It could also rescue non-top-ranked glycopeptide hits. Identification of glycopeptides is particularly susceptible to precursor mass errors for their low abundances, large masses, and glycans differing by 1 or 2 Da easily confused as isotopes. In addition, the XIC-centric strategy significantly reduced the problem of one XIC peak associated with multiple unique identifications, a source of quantitative irreproducibility. Taken together, the proposed approach can lead to improved identification and quantification accuracy and, ultimately, enhanced reproducibility in proteomic data analyses.

Decoding the glycoproteome: a new frontier for biomarker discovery in cancer.

Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.

High-throughput N-glycoproteomics with fast liquid chromatographic separation.

N-glycosylation is a common protein post translation modification, which has tremendous structure diversity and wide yet delicate regulation of protein structures and functions. Mass spectrometry-based N-glycoproteomics has become a state-of-the-art pipeline for both qualitative and quantitative characterization of N-glycosylation at the intact N-glycopeptide level, providing comprehensive information of peptide backbones, N-glycosites, monosaccharide compositions, sequence and linkage structures. For high-throughput analysis of large-cohort clinic samples, fast and high-performance separation is indispensable. Here we report our development of 1-h liquid chromatography gradient N-glycoproteomics method and accordingly optimized MS parameters. In the benchmark analysis of cancer and paracancerous tissue of hepatocellular carcinoma, 5,218 intact N-glycopeptides were identified, where 422 site- and structure-specific differential N-glycosylation on 145 N-glycoproteins was observed. The method, representing substantial increase of throughput, can be adopted for fast and efficient analysis of N-glycoproteomes at large scale.

Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS.

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

Cell surface glycoproteomics: deciphering glycoproteins through a unique analytical capture approach.

Cell surface proteins (CSPs) are often involved in various biological processes such as cell-cell interactions, immune responses, and molecular transport. The abnormal expression of CSP usually indicates the occurrence and development of human diseases. Most CSPs are glycosylated and have been explored as potential drug targets and disease biomarkers; however, efficient isolation of CSPs from intracellular proteins is difficult due to their low abundance and strong hydrophobicity. The comprehensive characterization of surface glycoproteins remains a great challenge and is often underrepresented in proteomics. In recent years, unprecedented progress has been made in the mass spectrometry analysis of surface proteins, and CSP capture methods and mass spectrometry have been greatly developed. In this article, we aim to give a comprehensive overview of innovative analytical methods that can enrich CSPs, including centrifugation-based separation, phase partitioning, adhesion-based capture of surface proteins, antibody or lectin affinity, and biotin-based chemical labeling. Surface glycoproteins are captured by chemical oxidation of glycans or click chemistry for carbohydrate metabolic labeling. These techniques offer a wide range of applications for studying the function of cell surface receptors and identifying markers for diagnostic and therapeutic development.

Protein glycosylation and glycoinformatics for novel biomarker discovery in neurodegenerative diseases.

Glycosylation is a common post-translational modification of brain proteins including cell surface adhesion molecules, synaptic proteins, receptors and channels, as well as intracellular proteins, with implications in brain development and functions. Using advanced state-of-the-art glycomics and glycoproteomics technologies in conjunction with glycoinformatics resources, characteristic glycosylation profiles in brain tissues are increasingly reported in the literature and growing evidence shows deregulation of glycosylation in central nervous system disorders, including aging associated neurodegenerative diseases. Glycan signatures characteristic of brain tissue are also frequently described in cerebrospinal fluid due to its enrichment in brain-derived molecules. A detailed structural analysis of brain and cerebrospinal fluid glycans collected in publications in healthy and neurodegenerative conditions was undertaken and data was compiled to create a browsable dedicated set in the GlyConnect database of glycoproteins (https://glyconnect.expasy.org/brain). The shared molecular composition of cerebrospinal fluid with brain enhances the likelihood of novel glycobiomarker discovery for neurodegeneration, which may aid in unveiling disease mechanisms, therefore, providing with novel therapeutic targets as well as diagnostic and progression monitoring tools.

Analysis of N-glycosylation protein of Kashin-Beck disease chondrocytes derived from induced pluripotent stem cells based on label-free strategies with LC-MS/MS.

We aimed to compare N-glycosylation proteins in Kashin-Beck disease (KBD) chondrocytes and normal chondrocytes derived from induced pluripotent stem cells (iPSCs). KBD and normal iPSCs were reprogrammed from human KBD and normal dermal fibroblasts, respectively. Subsequently, chondrocytes were differentiated from KBD and normal iPSCs separately. Immunofluorescence was utilized to assay the protein markers of iPSCs and chondrocytes. Differential N-glycosylation proteins were screened using label-free strategies with LC-MS/MS. Bioinformatics analyses were utilized to interpret the functions of differential N-glycosylation proteins. Immunofluorescence staining revealed that both KBD-iPSCs and normal-iPSCs strongly expressed pluripotency markers OCT4 and NANOG. Meanwhile, chondrocyte markers collagen II and SOX9 are presented in KBD-iPSC-chondrocytes and normal-iPSC-chondrocytes. We obtained 87 differential N-glycosylation sites which corresponded to 68 differential proteins, which were constructed into 1 cluster. We obtained collagen type I trimer and 9 other biological processes; polysaccharide binding and 9 other molecular functions; regulation of transcription by RNA polymerase II and 9 other cellular components from GO; the Pl3K-Akt signaling pathway and 9 other KEGG pathways; peroxisome and 7 other subcellular locations; and integrin alpha chain, C-terminal cytoplasmic region, conserved site and 9 other classifications of domain annotations, and 2 networks. FGFR3 and LRP1 are expressed at higher levels in KBD-iPSC-chondrocytes, while the expressions of COL2A1, TIMP1, UNC5B, NOG, LEPR, and ITGA1 were down-regulated in KBD-iPSC-chondrocytes. The differential expressions of these N-glycosylation proteins may lead to the abnormal function of KBD chondrocytes.

Association of activins, follistatins and inhibins with incident hip fracture in women with postmenopausal osteoporosis: a proof of concept, case-control study.

PURPOSE: The activins-follistatins-inhibins (AFI) hormonal system is considered to regulate muscle and bone mass. We aimed to evaluate AFI in postmenopausal women with an incident hip fracture. METHODS: In this post-hoc analysis of a hospital based case-control study, we evaluated circulating levels of the AFI system in postmenopausal women with a low-energy hip fracture admitted for fixation compared with postmenopausal women with osteoarthritis scheduled for arthroplasty. RESULTS: Circulating levels of follistatin (p = 0.008), FSTL3 (p = 0.013), activin B and AB (both p < 0.001), as well as activin AB/follistatin and activin AB/FSTL3 ratios (p = 0.008 and p = 0.029, respectively) were higher in patients than controls in unadjusted models. Differences for activins B and AB remained after adjustment for age and BMI (p = 0.006 and p = 0.009, respectively) and for FRAX-based risk for hip fracture (p = 0.008 and p = 0.012, respectively) but were lost when 25OHD was added to the regression models. CONCLUSIONS: Our data indicate no major changes in the AFI system in postmenopausal women at the time of hip fracture compared to postmenopausal women with osteoarthritis except for higher activin B and AB levels, whose significance, however, was lost when 25OHD was added to the adjustment models. CLINICAL TRIALS: Clinical Trials identifier: NCT04206618.

Integrative Proteomics and N-Glycoproteomics Analyses of Rheumatoid Arthritis Synovium Reveal Immune-Associated Glycopeptides.

Rheumatoid arthritis (RA) is a typical autoimmune disease characterized by synovial inflammation, synovial tissue hyperplasia, and destruction of bone and cartilage. Protein glycosylation plays key roles in the pathogenesis of RA but in-depth glycoproteomics analysis of synovial tissues is still lacking. Here, by using a strategy to quantify intact N-glycopeptides, we identified 1260 intact N-glycopeptides from 481 N-glycosites on 334 glycoproteins in RA synovium. Bioinformatics analysis revealed that the hyper-glycosylated proteins in RA were closely linked to immune responses. By using DNASTAR software, we identified 20 N-glycopeptides whose prototype peptides were highly immunogenic. We next calculated the enrichment scores of nine types of immune cells using specific gene sets from public single-cell transcriptomics data of RA and revealed that the N-glycosylation levels at some sites, such as IGSF10_N2147, MOXD2P_N404, and PTCH2_N812, were significantly correlated with the enrichment scores of certain immune cell types. Furthermore, we showed that aberrant N-glycosylation in the RA synovium was related to increased expression of glycosylation enzymes. Collectively, this work presents, for the first time, the N-glycoproteome of RA synovium and describes immune-associated glycosylation, providing novel insights into RA pathogenesis.

High-sensitivity biosensor based on SERS integrated with dendrimer-assisted boronic acid-functionalized magnetic nanoparticles for IL-6 detection in human serum.

High-sensitivity quantitative analysis of sepsis disease markers in circulating blood is essential for sepsis early diagnosis, rapid stratification, and interventional treatment. Herein, a high-sensitivity biosensor combining surface-enhanced Raman spectroscopy (SERS) and functionalized magnetic materials was developed to quantitatively detect interleukin-6 (IL-6), a glycoprotein disease marker closely related to sepsis. First, boronic acid-functionalized magnetic nanomaterials with high adsorption performance were synthesized by utilizing the branched polyethyleneimine to provide many binding sites for boronic acid. Under antibody-free conditions, dendrimer-assisted boronic acid-functionalized magnetic nanomaterials selectively capture glycoproteins in complex biological samples as bio-capture element. Then, a core-shell bimetallic material with plenty of 'hot spots' was designed and synthesized as the enhancement substrate. The 4-Mercaptobenzonitrile (4-MP) with a characteristic peak at 2224 cm-1(Raman-silent region) was embedded as the Raman reporter to form a SERS immune probe with highly efficient electromagnetic enhancement effect, achieving specific recognition and high-sensitivity detection of IL-6 on bio-capture elements. Using this strategy for quantitative analysis of IL-6, a wide detection range (0.5-5000 pg ml-1) and a low detection limit (0.453 pg ml-1) were obtained. Moreover, this method exhibited excellent detection performance for IL-6 in human serum samples, demonstrating its potential promise in screening clinically relevant diseases. The biosensor presented here not only provides a novel and universally applicable sensing strategy for the enrichment and detection of trace glycoprotein disease markers, but also the application of a portable Raman spectrometer provides a more reliable experimental basis for the diagnosis and treatment of major diseases in the clinic or remote and deprived areas.

High-resolution mass spectrometry for glycoproteomics.

Tweetable abstract Bottom-up glycoproteomics combined with top-down strategy allows direct analysis of glycoform-mapped glycosylation and its glycans by high-resolution mass spectrometry.

N-Linked Glycoproteome Analysis of Diosorea alata Tuber Shows Atypical Glycosylation and Indicates Central Role of Glycosylated Proteins in Tuber Maturation.

Glycosylation is an important post translational modification in plants. First analysis of N-linked glycosylated proteins of Dioscorea alata using Concanavalin A lectin affinity chromatography enrichment coupled with label free quantification is presented. In total, 114 enriched glycoproteins were detected. Signal P and sub-cellular localization showed 42.2% of proteins to be secretory. These included peroxidases, endochitinases, calreticulin, calnexin, thaumatins and lipid transfer proteins. Gene Ontology and MapMan analysis predicted the enriched glycoproteins to be involved in processes essential for tuber maturation namely: signal transduction, lignification, protein trafficking, endoplasmic reticulum quality control and cell wall remodeling. This was supported by biochemical validation of the essential glycoproteins. Interestingly, out of the two dioscorin isoforms, Dio B was the only N-glycosylated form. In silico analysis showed O-glycosylation sites in the other form, Dio A suggesting its similarity with sporamin, the storage protein of sweet potato. Absence of signal peptide in Dio B and the presence of non-canonical motif hints towards its atypical glycosylation. The analysis revealed that N-glycosylation of Dio B isoform maintains the activities associated with Dioscorin at maturity and provides an overview of protein N-glycosylation, enriching the glycoproteome database of plants especially tubers.

Characterization of sow milk N-linked glycoproteome over the course of lactation.

Milk proteins serve as nutrition and affect neonate development and immunity through their bioactivity. Post-translational modifications of proteins affect their bioactivity. Glycosylation is the attachment of sugar moieties to proteins, with attachment of glycans to asparagine indicated as N-linked glycosylation. Our objective was to characterize N-linked glycosylated proteins in homogenate swine milk samples collected from sows (n = 5/6) during farrowing to represent colostrum and on days 3 and 14 post-farrowing to represent transitional and mature milk, respectively. Glycopeptides were isolated with lectin-based extraction and treated with Peptide N-glycosidase F (PNGase F) to identify N-linked glycosylation sites. Purified glycopeptides were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant software was used to align spectra to Sus scrofa Uniport database to identify proteins and measure their relative abundances. Analysis of variance and Welch's t-test analysis identified glycoproteins differentially abundant between colostrum, transitional, and mature milk (false discovery rate <0.05). Shotgun proteome analysis identified 545 N-linked and glutamine, Q, -linked, glycosylation (P > 0.75 for deamidation) sites on 220 glycoproteins in sow milk. Glycoproteins were found across all three phases of swine milk production and varied by number of glycosylation sites (1-14) and in abundance and distribution between colostrum, transitional, and mature milk. Polymeric immunoglobulin receptor was the most glycosylated protein with 14 sites identified. Also highly glycosylated were casein and mucin proteins. These data are described and the relevance of glycosylated milk proteins in neonate development, such as protection against pathogens, is discussed.

Milk is essential for healthy growth and development of neonates, with proteins in milk serving as key nutrients and regulators of these processes. Protein activity is affected by modifications made to their structure including the addition of sugar groups called glycans. Here we present the characterization of sow milk proteins modification with glycan groups on asparagine and glutamine amino acids in colostrum, transitional, and mature milk of pigs. We found 220 high confidences (found in at least two sows on one day) glycoproteins, and that the abundance of glycosylated proteins varied by stage of milk production and number of glycosylated sites.

Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

Methods for quantification of glycopeptides by liquid separation and mass spectrometry.

Recent advances in analytical techniques provide the opportunity to quantify even low-abundance glycopeptides derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Herein, we discuss the sample preparation procedures and the mass spectrometry (MS) strategies that have facilitated glycopeptide quantification, as well as the standards used for glycopeptide quantification. For sample preparation, various glycopeptide enrichment methods are summarized including the columns used for glycopeptide separation in liquid chromatography separation. For MS analysis strategies, MS1 level-based quantification and MS2 level-based quantification are described, either with or without labeling, where we have covered isotope labeling, TMT/iTRAQ labeling, data dependent acquisition, data independent acquisition, multiple reaction monitoring, and parallel reaction monitoring. The strengths and weaknesses of these methods are compared, particularly those associated with the figures of merit that are important for clinical biomarker studies and the pathological and functional studies of glycoproteins in various diseases. Possible future developments for glycopeptide quantification are discussed.

Partial Characterization of Lectins Purified from the Surco and Vara (Furrow and Rod) Varieties of Black Phaseolus vulgaris.

As they manifest specifically and reversibly, lectins are proteins or glycoproteins with the characteristic of agglutinating erythrocytes. Given that grain legume lectins can represent 10% of protein content and can have various biological functions, they are extensively studied. The objective of this work was to purify and partially characterize the lectins of Phaseolus vulgaris black, var surco and vara (LBBS and LBBV). Both lectin types were purified by affinity chromatography on stroma matrix, which agglutinated human erythrocytes type A, B, and O, as well as rabbit, hamster, pig, and chicken erythrocytes. Native-PAGE was employed for molecular mass determination, yielding 109.36 and 112.68 kDa for BBS and BBV, respectively. Further analyses revealed that these lectins are tetrameric glycoproteins that require Ca+2, Mn+2 and Mg+2 ions for exhibiting their hemagglutinating function, which can be inhibited by fetuin. Moreover, optimal pH was established for both lectins (10.5 for LBBS and 7-9 for LBBV), while their activity was temperature-dependent and ceased above 70 °C. Finally, the observed differences in the biochemical characteristics and bioactive functions were ascribed to the different physiological characteristics of each seed, as well as the protein itself.