Degree Of Glycosylation Research Articles

Importance of glycosylation for SARS-CoV-2 virus, antibodies and COVID-19

Summary In addition to the known endemic respiratory viruses, the pandemic SARS-CoV-2 has been added, with terrible effects on the population worldwide. This virus, like many others, has a glycoprotein structure the so-called spike protein, which interacts with the ACE-2 receptor as a cellular entry gate and thus initiates the infection. Just as this glycosylation causes infectiousness, the effect of the protective antibodies is also determined by their degree of glycosylation. In particular, the so-called Fc part of the immunoglobulins plays an important role. The type of bound sugar such as fucose, mannose, sialic acid, etc. determines, among other things, which reactions are triggered by binding to immune cells such as NK cells to their existing Fc receptors. These include antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular trogocytosis (ADCT) and antibody-dependent cellular complement deposition (ADCD). In addition to protective phagocytosis, these reactions can trigger the antibody-dependent enhancement (ADE) of the infection and also the dreaded cytokine storm in COVID-19 patients. The functional diversification of IgGs by Fc glycosylation can be determined and tracked both after vaccination and during the disease.

Structural characteristics and acid-induced emulsion gel properties of heated soy protein isolate–soy oligosaccharide glycation conjugates

Glycation conjugates were prepared by heating the mixed dispersions of heat-denatured soy protein isolate (HSPI) and soy oligosaccharide (SOS) at 90 °C for 30, 60, 90 and 120 min. The structural characteristics and acid-induced emulsion gel properties of HSPI–SOS conjugates were investigated. The results of degree of graft, pH, intermediates and browning intensity showed that the degree of glycosylation gradually increased with the extension of reaction time, but it decreased at 120 min of reaction. The changes of the absorption peaks in infrared spectra confirmed that HSPI was covalently linked with SOS to form HSPI–SOS conjugates through Maillard reaction. The glycosylation of HSPI with SOS resulted in the transformation form α-type to β-type secondary structure, the reduction in free sulfhydryl groups, and the enhancement of surface hydrophobicity. These structural changes of HSPI–SOS conjugates could influence their glucono-δ-lactone-induced emulsion gel properties. Although the glycosylation decreased the storage modulus of HSPI emulsion gels, the 60 min glycated conjugates accelerated the gelation rate of the emulsions. The conjugate emulsion gels had higher gel hardness and water holding capacity, which exhibited more compact microstructure with smaller and more uniform emulsified oil droplets. Moreover, the glycosylation of HSPI with SOS for 60 min improved the thermal stability of conjugate emulsion gels. Therefore, the structure of HSPI–SOS conjugates can be modified by controlling the degree of glycosylation to improve the physicochemical properties and stability of the emulsion gels.

Influences of the different chemical components in soybean soluble polysaccharide on the emulsifying performance of its conjugates formed with soybean protein isolate

SummaryThis work aimed to elucidate the relationship between the chemical components of soybean soluble polysaccharide (SSPS) and its Maillard reaction with soybean protein isolate (SPI). Enzymatic hydrolysis was used to specifically cleave the chemical components of SSPS. Neutral sugar side chain regions of SSPS were easier to conjugate with SPI than the backbone containing galacturonic acid. The modified SSPS with higher content of arabinose showed the highest degree of glycosylation (46.8%). SSPS‐SPI conjugates exhibited lower interfacial tension than SPI, fabricating emulsion with smaller droplets size and better emulsion stability. The improved emulsifying properties could be ascribed to two aspects: (i) a higher percentage of adsorbed protein at the interface to cover more oil droplet surface; (ii) thicker adsorbed layer thickness onto the droplet surface to provide stronger steric‐hindrance effect to prevent droplet coalescence.

Effect of Maillard reaction conditions on the gelation and thermal stability of whey protein isolate/d-tagatose conjugates

The Maillard reaction (MR) was used as a natural modification method to improve the functionality of proteins by covalent interaction with monosaccharides. This study aimed to prepare whey protein isolate (WPI)/d-Tagatose (DT) conjugates by dry heating treatment to investigate the effect of different MR conditions on the structural changes of WPI and its effect on the WPI/DT conjugates gelation and thermal stability. Covalent interactions between WPI and DT were detected using multi-spectral methods. The MR conditions significantly affected the degree of glycosylation, structure, and molecular weight of the WPI/DT conjugates (P < 0.05). The MR changed the WPI/DT conjugates gel properties and thermal stability, where the WPI/DT 1:1–1 gel properties were stronger than those of WPI-1 and the heating deflection temperature after glycosylation was increased to 163.55 °C (P < 0.05). This study provides a theoretical basis for the application of DT as food ingredient in the development of WPI functional properties by MR.

Garlic essential oil emulsions stabilized by microwave dry-heating induced protein-pectin conjugates and their application in controlling nitrite content in prepared vegetable dishes

The main objective of this study was to assess whether microwave dry-heating (MDH) can shorten the conjugation time and achieve similar level of glycosylation between soy protein isolate (SPI) and citrus pectin (CP) obtained using traditional dry-heating (TDH). It also involved the application of SPI-CP conjugates produced by MDH (SPI-CP-MDH) as an emulsifier of garlic essential oil (GEO) emulsions and used these emulsions to control nitrite content of prepared vegetable dishes (PVDs). At a power density of 1.5 W/g, MDH shortened the conjugation time by 25% and achieved higher glycosylation degree, compared to TDH. SPI-CP-MDH conjugates exhibited comparable emulsifying and emulsion stabilizing characteristics on GEO emulsions, compared with SPI-CP-TDH conjugates. The ability of SPI-CP-MDH conjugates to inhibit the formation of nitrite in PVDs was higher than that of SPI-CP-TDH conjugates. The control of nitrite formation was attributable to the inhibition of nitrite-producing bacteria through antioxidant and anti-light properties of emulsions rather than reducing enzyme activity associated with nitrite in plant cells. MDH can be an alternative to TDH for producing SPI-CP conjugates. GEO emulsions stabilized by SPI-CP-MDH conjugates can potentially be a preferred option to control nitrite content in PVDs.

Structural and functional properties of sodium caseinate glycosylated by dextran aldehyde

Glycosylation is an effective method of protein modification which can improve the functional properties of proteins. Traditional protein glycosylation methods are typically limited by slow reaction rates. A method for improving the efficiency of protein glycosylation from different degrees of dextran oxidation with periodate, to obtain dextran aldehydes, is presented. Increased molar ratio between periodate and glucose unit in dextran led to dextran aldehyde with higher aldehyde group content and reduced molecular weight. This is due to an increased oxidation, which increases the degree of glycosylation by 43.22%. Compared with dextran glycosylated products, the emulsifying activity, stability indices and foaming capacity of dextran aldehyde glycosylated products increased by 30.8, 301 and 50%, respectively, with poor foam stability. Dextran aldehyde-modified sodium caseinate presented lower digestibility and could maintain better stability against salt ions and heat treatment. The polysaccharide aldehyde showed great potential for expanding protein modification methods to promote its industrialization.

Changes in partial properties of glycosylated egg white powder during storage.

Glycosylation is an effective method to modify protein. However, there is a lack of research on the property changes of glycosylated protein during storage. In the present study, the changes in the physicochemical, functional, and structural properties of xylo-oligosaccharide (XOS) glycosylated egg white powder (EWP) (XOS-EWP conjugates) prepared with different glycosylation conditions (XOS/EWP ratio and reaction time) were investigated when stored at 25 °C and 60% relative humidity. In the 12 weeks of storage, the degree of grafting, browning, and the formation of Maillard reaction products of XOS-EWP conjugates increased. The increase in XOS/EWP ratio and reaction time led to an increase in protein aggregation, though a decrease in solubility, due to increased degree of glycosylation and structural changes. Furthermore, improved gel hardness of XOS-EWP conjugates deteriorated, while improved emulsification ability was kept stable during storage. For the sample with a lower XOS/EWP ratio and reaction time, the gel hardness and emulsifying properties underwent little or no deterioration even improving during storage. The results could be attributed to the limited degree of glycosylation, further unfolding of the protein structure, increased surface hydrophobicity of protein, and improved thermal characteristics. During storage, the Maillard reaction would continue to occur in the glycosylated EWP, further affecting the performance of modified EWP. Modified EWP prepared under different glycosylation conditions performed differently during storage. Modified EWP with a larger XOS/EWP ratio and reaction time meant it was harder to maintain good performance. Modified EWP with a smaller XOS/EWP ratio and reaction time changed significantly to better performances. © 2022 Society of Chemical Industry.

Characterization and immunogenicity of SARS-CoV-2 spike proteins with varied glycosylation

The ongoing coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drastically changed our way of life and continues to have an unmitigated socioeconomic impact across the globe. Research into potential vaccine design and production is focused on the spike (S) protein of the virus, which is critical for virus entry into host cells. Yet, whether the degree of glycosylation in the S protein is associated with vaccine efficacy remains unclear. Here, we first optimized the expression of the S protein in mammalian cells. While we found no significant discrepancy in purity, homogeneity, or receptor binding ability among S proteins derived from 293F cells (referred to as 293F S-2P), 293S GnTI- cells (defective in N-acetylglucosaminyl transferase I enzyme; 293S S-2P), or TN-5B1-4 insect cells (Bac S-2P), there was significant variation in the glycosylation patterns and thermal stability of the proteins. Compared with the partially glycosylated 293S S-2P or Bac S-2P, the fully glycosylated 293F S-2P exhibited higher binding reactivity to convalescent sera. In addition, 293F S-2P induced higher IgG and neutralizing antibody titres than 293S or Bac S-2P in mice. Furthermore, a prime-boost-boost regimen, using a combined immunization of S-2P proteins with various degrees of glycosylation, elicited a more robust neutralizing antibody response than a single S-2P alone. Collectively, this study provides insight into ways to design a more effective SARS-CoV-2 immunogen.

Biochemical characterisation of glycosylated and deglycosylated forms of phytase from Cronobacter turicensis expressed in Pichia pastoris

Phytases are valuable industrial enzymes widely used in animal feed production, and when expressed in yeast, they undergo glycosylation. Herein, heterologous expression in Pichia pastoris and biochemical characterisation of glycosylated and deglycosylated forms of a novel phytase from Cronobacter turicensis belonging to the histidine acid phosphatase family were successfully carried out. Mutants with deleted N-glycosylation sites (N136, N171, and N202) were constructed by site-directed mutagenesis and characterised. Deglycosylation greatly changed the resistance of phytase to proteolysis, acidic pH, and temperature, but did not significantly affect other biochemical properties. High specific activity (1705 U/mg), Km (173 μM), and Vmax (2778 μmol min−1 mg−1), as well as the optimum temperature (50 °C) and pH (4.5) of the enzyme did not depend on the degree of glycosylation. The degree of change in resistance of phytase depended on the position of the N-glycosylation site on the protein globule. The removal of glycan at the N202 site located in the highly flexible region of the protein had the greatest effect on the enzyme characteristics and led to a decrease in resistance of phytase to pepsin and trypsin by 73% and 87%, respectively. Conversely, the glycosylated enzyme exhibited strong resistance to pepsin, trypsin, and acidic pH, and increased thermostability. It has been shown that N-glycosylation occurring in P. pastoris served as a powerful tool for improving the biochemical properties of recombinant phytase and made it promising for use as an animal feed additive.

A multiplexed parallel reaction monitoring assay to monitor bovine pregnancy-associated glycoproteins throughout pregnancy and after gestation.

Bovine pregnancy-associated glycoproteins (boPAGs) are extensively glycosylated secretory proteins of trophoblast cells. Roughly 20 different boPAG members are known but their distribution patterns and degree of glycosylation during pregnancy are not well characterized. The objective of the present study was the development of a parallel reaction monitoring-based assay for the profiling of different boPAGs during pregnancy and after gestation. Furthermore, we investigated the effects of N-glycosylation on our analytical results. BoPAGs were purified from cotyledons of four different pregnancy stages. The assay detects 25 proteotypic peptides from 18 boPAGs in a single run. The highest abundances were found for boPAG 1 in both, glycosylated and deglycosylated samples. Strongest effects of glycosylation were detected during mid and late pregnancy as well as in afterbirth samples. Furthermore, we identified different boPAG-clusters based on the observed relative protein abundances between glycosylated and deglycosylated samples. A linkage between the impact of glycosylation and potential N-glycosylation sites or phylogenetic relation was not detected. In conclusion, the newly developed parallel reaction monitoring-based assay enables for the first time a comprehensive semi-quantitative profiling of 18 different boPAGs during pregnancy and post-partum on protein level, thereby investigating the influence of glycosylation. The results of this study provide new and important starting points to address further research on boPAGs to better understand their physiological role during pregnancy and for the development of new pregnancy detection tests.

Optimization of enzymatic soy protein isolate-glucosamine conjugates to improve the freeze-thaw stability of emulsion.

Using transglutaminase (TGase) is a new method to improve protein properties in order to promote protein glycosylation. This article mainly studies soy protein isolate (SPI) and glucosamine to improve the freeze-thaw stability of emulsion under the action of TGase. The degree of glycosylation was studied by the content of free amino groups and the degree of conjugation. The optimal conditions for preparing soy protein isolate-glucosamine (SPI-G) conjugate were determined by a response surface optimization model based on single-factor experiments using the creaming index of the emulsion after the first freeze-thaw cycle as the response value. The results showed that the emulsion had the lowest creaming index when the conditions of protein concentration was 20 g L-1 , mass ratio of SPI-G was 5:3 (w/w), enzyme addition amount was 10U g-1 , and reaction time was 2h. The optimized modified product was measured for the creaming index after the first freeze-thaw cycle. It was found that the creaming index of the modified product SPI-G after the first freeze-thaw cycle was 9.02%, which was less than and close to the optimized model predicted value. The creaming index and optical microscopy results after three freeze-thaw cycles confirmed that the freeze-thaw stability of the SPI-G samples was significantly enhanced after optimization of the response surface model. It showed that glycosylation promoted by TGase could improve the freeze-thaw stability of SPI emulsion, thereby broadening the application of SPI in food. © 2022 Society of Chemical Industry.

Effect of ultrasound on the glycosylation reaction of pea protein isolate–arabinose: Structure and emulsifying properties

This study investigated the effects of different ultrasonic power and ultrasonic time on the structure and emulsifying properties of pea protein isolate (PPI)–arabinose conjugates. An examination of the absorbance and color development of PPI-d-arabinose (Ara) conjugates found that compared with traditional heating, the degree of glycosylation of protein reached the maximum when the ultrasonic treatment power was 150 and the treatment time was 30 min. Structural analysis revealed that the content of disordered structures (β-turn and random coil) of the protein conjugates increased, the maximum emission wavelength of the fluorescence spectrum was red-shifted, and the UV second-order derivative values decreased. The protein structure unfolded, exposing more hydrophobic groups on the molecular surface. Ultrasonic treatment improved the emulsification of protein conjugates. The emulsifying activity index (EAI) increased to 19.7 and 19.3 m2/g, and the emulsifying stability index (ESI) also increased. The contact angle and zeta potential also demonstrate that ultrasonic power has a positive effect on emulsion stability. Based on examining the thermal stability of the emulsion, the ultrasonic treatment increased the thermal denaturation resistance of the protein. This result confirms that mild sonication can increase the degree of glycosylation reaction and improve the emulsification properties of protein–Ara conjugates, providing a theoretical basis for developing foods with excellent emulsification properties.

The Influence of Clusterin Glycosylation Variability on Selected Pathophysiological Processes in the Human Body.

The present review gathers together the most important information about variability in clusterin molecular structure, its profile, and the degree of glycosylation occurring in human tissues and body fluids in the context of the utility of these characteristics as potential diagnostic biomarkers of selected pathophysiological conditions. The carbohydrate part of clusterin plays a crucial role in many biological processes such as endocytosis and apoptosis. Many pathologies associated with neurodegeneration, carcinogenesis, metabolic diseases, and civilizational diseases (e.g., cardiovascular incidents and male infertility) have been described as causes of homeostasis disturbance, in which the glycan part of clusterin plays a very important role. The results of the discussed studies suggest that glycoproteomic analysis of clusterin may help differentiate the severity of hippocampal atrophy, detect the causes of infertility with an immune background, and monitor the development of cancer. Understanding the mechanism of clusterin (CLU) action and its binding epitopes may enable to indicate new therapeutic goals. The carbohydrate part of clusterin is considered necessary to maintain its proper molecular conformation, structural stability, and proper systemic and/or local biological activity. Taking into account the wide spectrum of CLU action and its participation in many processes in the human body, further studies on clusterin glycosylation variability are needed to better understand the molecular mechanisms of many pathophysiological conditions. They can also provide the opportunity to find new biomarkers and enrich the panel of diagnostic parameters for diseases that still pose a challenge for modern medicine.

Inhibition of highland barley bran-derived carbon dots on the formation of advanced glycation end products

Advanced glycation end products (AGEs) are among the series factors that cause the pathology of neurodegenerative diseases. Carbon dots (CDs) with low toxicity, tunable surface chemistry and biocompatibility have been used for biological inhibition and as anticancer agents. In this study, CDs derived from highland barley bran (HBB-CDs) were used to inhibit the formation of AGEs in vitro. The possible inhibitory mechanism was attributed to the decreased number of glycosylation sites in glycosylated β-casein from four and six to three and four in the presence of the HBB-CDs, which was revealed by HPLC–Orbitrap tandem mass spectrometry at the molecular level. Furthermore, the glycosylation degree of each glycosylation site was commanded, and the double saccharification capacity of K167 and K214 was changed. Strong inhibition of AGEs formation in the presence of elevated HBB-CDs suggested that CDs are potential anti-glycation agents and food additives against glycation-associated diseases and food processing.

Effects of detoxification process on toxicity and foreign protein of tetanus toxoid and diphtheria toxoid

Formaldehyde detoxification is a process for converting tetanus toxin (TT) and diphtheria toxin (DT) into tetanus toxoid (TTd) and diphtheria toxoid (DTd), respectively. The mechanism of this detoxification process has been investigated by several previous studies based on lab-scale toxoids. To obtain greater insights of the effects induced by formaldehyde, industrial TTd and DTd batches obtained from different detoxification processes were studied in this work. Using liquid chromatography-mass spectrometry (LC-MS), 15 and 20 repeatable formaldehyde-induced modification sites of TTd and DTd were identified, respectively. Toxoid which had a higher formaldehyde-induced modification rate observed by LC-MS, also had larger bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Aggregates which were observed on size exclusion chromatogram (SEC) were confirmed by SDS-PAGE and LC-MS. Formaldehyde detoxification also led to a decrease of isoelectric point (pI) values and an increase of retention on weak anion exchange (WAX) column. Specific toxicity tests were conducted to evaluate toxicity of the TTd and DTd samples obtained with different detoxification conditions. Results from the specific toxicity tests showed that all toxoids used in this study were qualified, including toxoids obtained from mild and drastic detoxification conditions. However, obtained from mild detoxification conditions had less aggregates and may lead to a higher degree of glycosylation in conjugate vaccines than the ones obtained from drastic detoxification conditions. Thus, we suggest that mild detoxification conditions should be used to obtain TTd and DTd. Furthermore, as well as studying the formaldehyde-induced modifications and toxicity in TTd and DTd, the effects of the detoxification process on foreign proteins were also investigated. An increase in foreign proteins were observed in the aggregate than in the monomer of the toxoids. Additionally, some foreign proteins in the monomer of the toxins transferred to the aggregate of toxoids due to the formation of cross-linking. To eliminate the risk of cross-linking foreign proteins to toxoids in vaccination programs, a purification process is necessary before the detoxification process and/or the use of toxoids in vaccines.

Effect of ball milling-assisted glycosylation modification on the structure and foaming property of egg white protein.

The effect of different glycosylation degrees on molecular structure and foaming property of egg white protein (EWP) was investigated using ball milling-assisted glycosylation. The results showed the foaming ability (FA) and foam stability (FS) of EWP improved when the degree of glycosylation was increased. In particular, FA of ball milling-assisted glycosylation of EWP enhanced by 39.9% and 28.8%, and the FS increased by 28.7% and 24.0% compared with EWP and ball milling egg white protein (BE) at 150min of reaction. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis could reflect the grafting degree of EWP and glucose molecules from the side. When EWP was fully grafted with glucose, endogenous fluorescence and free sulfhydryl groups indicated that tertiary structure of EWP was depolymerized, and Fourier transform infrared spectroscopy showed the secondary structure tended to change from order to disorder. The results of this study indicated that ball milling-assisted glycosylation modification was a practical method to improve the foaming property of EWP. PRACTICAL APPLICATION: EWP has great FA and FS, making it indispensable in the baking industry. In this study, ball milling-assisted glycosylation was used to improve the foaming property of EWP, and the molecular structure of EWP with different degrees of glycosylation was fully resolved. The results demonstrated that ball milling, as a physical pretreatment, can fully unfold the structure of EWP. When sugar molecules were fully grafted, the particle size of EWP reduced, solubility increased, and the stability of system improved, thus enhancing the foaming property of EWP. The results can provide theoretical basis for improving the foaming property of EWP and provide a reference value for its industrial application.

Evidence for the Existence of Two Prolactin Isoforms in the Developing Pituitary Gland of the Goose (Anser cygnoides)

Compared to Galliformes such as chicken and turkey, very little is known about the existence and expression of isoforms of prolactin (PRL) in the pituitary glands of Anseriformes. In this study, by generating a rabbit-anti-goose (Anser cygnoides) PRL polyclonal antibody, we analysed the expression patterns of goose PRL isoforms in the embryonic and post-hatch development of the pituitary gland. Our results showed that two immunoreactive bands with molecular weights of about 23 and 26 kDa were detected using the Western blot technique, corresponding to the non-glycosylated (NG-) and the glycosylated (G-) isoform of PRL, respectively. The protein levels of the total PRL in a goose increased gradually from the embryonic day (ED) 22 to the post-hatch day (PD) 28, with a non-significant decrease on PD6. Furthermore, the percentage of G-PRL in the pituitary gland of the goose fluctuated from about 30.3% to 54.7% throughout the embryonic and post-hatch development. At the mRNA level, the expression of PRL increased steadily during the development and reached the highest levels on PD12, but later showed a non-significant decrease on PD28. The inconsistent expression patterns between the PRL mRNA and protein during the stages from PD6 to PD28 indicated that the PRL gene expression involves both transcriptional and post-translational regulation. Taken together, our data unequivocally demonstrated the existence of NG- and G-PRL in the pituitary gland of a goose and that the expression of the total PRL as well as the percentage of G-PRL significantly changed during embryonic and post-hatch development, indicating that the versatile biological functions of PRL during the ontogenesis of a goose could be closely related to changes in both its total expression and the degree of glycosylation in the pituitary gland.

Multi-approach LC-MS methods for the characterization of species-specific attributes of monoclonal antibodies from plants

In this work, an analytical platform based on the use of chromatography and mass spectrometry (MS), has been applied to the characterization of Rituximab (RTX) obtained from two plant expression systems (rice and tobacco) in comparison to the mammalian cell-derived reference monoclonal antibody (mAb). Different chromatographic approaches, hyphenated to high resolution MS (HRMS), were applied to RTX structural investigation both at middle- and peptide level. In particular, cation exchange chromatography (CEX), size exclusion chromatography (SEC), reversed phase (RPLC) and hydrophilic interaction liquid chromatographic (HILIC) methods were developed and applied on intact mAbs, IdeS-, and trypsin digests in order to address critical attributes such as primary structure, glycan composition, species-related heterogeneity, glycosylation degree, charge variants, aggregation tendency and enzymatic stability. All the collected data highlight the features and criticalities of each production approach. Production in rice results in a heterogeneous but stable product over time, suggesting the absence of proteases in seeds; while tobacco expression system leads to more homogeneous glycosylation, but protein stability seems to be a critical issue probably due to the presence of proteases. This analytical strategy represents a robust support to scientists in the selection and optimization of the best plant expression system to produce recombinant humanized mAbs.

A comparison between analytical approaches for molecular weight estimation of proteins with variable levels of glycosylation.

Biotherapeutics, such as mAbs and fusion proteins, are a major and rapidly growing class of pharmaceuticals. Majority of the biopharmaceuticals are glycoproteins, wherein about 1 to 30% of their molecular weight (MW) are contributed by the glycans. Determination of MW of heavily glycosylated proteins, such as Fc-fusion proteins, is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation influences the expected size of the glycoprotein, which leads to disproportionate MW estimation, in size-dependent methods. Hence, in this study, we have demonstrated the advantages and limitations of four widely used MW estimation techniques for three proteins having varying levels of glycosylation. It was proven that glycosylation had least impact on MW determination by SEC-MALS and SV-AUC. However, MW estimation by LC-MS and SDS-PAGE was extensively hampered by the degree of glycosylation. It is, thus, essential to consider the structural characteristics of proteins while selecting a technique for determining their MW.

Micro-Fourier-transform infrared reflectance spectroscopy as tool for probing IgG glycosylation in COVID-19 patients

It has been reported that patients diagnosed with COVID-19 become critically ill primarily around the time of activation of the adaptive immune response. However the role of antibodies in the worsening of disease is not obvious. Higher titers of anti-spike immunoglobulin IgG1 associated with low fucosylation of the antibody Fc tail have been associated to excessive inflammatory response. In contrast it has been also reported that NP-, S-, RBD- specific IgA, IgG, and IgM are not associated with SARS-CoV-2 viral load, indicating that there is no obvious correlation between antibody response and viral antigen detection. In the present work the micro-Fourier-transform infrared reflectance spectroscopy (micro-FTIR) was employed to investigate blood serum samples of healthy and COVID-19-ill (mild or oligosymptomatic) individuals (82 healthcare workers volunteers in “Instituto de Infectologia Emilio Ribas”, São Paulo, Brazil). The molecular-level-sensitive, multiplexing quantitative and qualitative FTIR data probed on 1 µL of dried biofluid was compared to signal-to-cutoff index of chemiluminescent immunoassays CLIA and ELISA (IgG antibodies against SARS-CoV-2). Our main result indicated that 1702–1785 hbox {cm}^{-1} spectral window (carbonyl C=O vibration) is a spectral marker of the degree of IgG glycosylation, allowing to probe distinctive sub-populations of COVID-19 patients, depending on their degree of severity. The specificity was 87.5 % while the detection rate of true positive was 100%. The computed area under the receiver operating curve was equivalent to CLIA, ELISA and other ATR-FTIR methods (>0.85). In summary, overall discrimination of healthy and COVID-19 individuals and severity prediction as well could be potentially implemented using micro-FTIR reflectance spectroscopy on blood serum samples. Considering the minimal and reagent-free sample preparation procedures combined to fast (few minutes) outcome of FTIR we can state that this technology is suitable for fast screening of immune response of individuals with COVID-19. It would be an important tool in prospective studies, helping investigate the physiology of the asymptomatic, oligosymptomatic, or severe individuals and measure the extension of infection dissemination in patients.