Differential expression of N-glycopeptides derived from serum glycoproteins in mild cognitive impairment (MCI) patients.

Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities with the aging of individuals, such as language or visual/spatial comprehension. MCI is considered a prodromal phase of more complicated neurodegenerative diseases such as Alzheimer's. Therefore, accurate diagnosis and better understanding of the disease prognosis will facilitate prevention of neurodegeneration. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of the serum N-glycoproteome expression could represent an essential contributor to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using less invasive procedures. In this approach, we identified N-glycopeptides with different expressions between healthy and MCI patients from serum glycoproteins. Seven of the N-glycopeptides showed outstanding AUC values, among them the antithrombin-III Asn224 + 4-5-0-2 with an AUC value of 1.00 and a p value of 0.0004. According to proteomics and ingenuity pathway analysis (IPA), our data is in line with recent publications, and the glycoproteins carrying the identified N-sites play an important role in neurodegeneration.

O-Glycome Profiling of Breast Cancer Cell Lines to Understand Breast Cancer Brain Metastasis.

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.

Analysis of Native and Permethylated N-Glycan Isomers Using MGC-LC-MS Techniques.

Glycosylation is an important post-translational modification that affects many critical cellular functions such as adhesion, signaling, protein stability, and function, among others. Abnormal glycosylation has been linked to many diseases. As such, the investigation of glycans and their roles in disease pathway and progression is important. Glycan analysis can be challenging, however, due to such factors as the heterogeneity of glycans and isomers as well as the poor ionization efficiency provided by mass spectrometry analyses. This chapter presents efficient methods that overcome these and other challenges for the analysis of native and permethylated N-glycan isomers in biological samples. Instructions regarding the packing of the MGC column, the N-glycan sample prep, and the LC-MS conditions are also provided.

Metabolomic Changes in Rat Serum after Chronic Exposure to Glyphosate-Based Herbicide.

Glyphosate-based herbicides (GBHs) have gained extensive popularity in recent decades. For many years, glyphosate has been regarded as harmless or minimally toxic to mammals due to the absence of its primary target, the shikimic acid pathway in humans. Nonetheless, mounting evidence suggests that glyphosate may cause adverse health effects in humans via other mechanisms. In this study, we described the metabolomic changes in the serum of experimental rats exposed to chronic GBH using the highly sensitive LC-MS/MS technique. We investigated the possible relationship between chronic exposure to GBH and neurological disorders. Our findings suggest that chronic exposure to GBH can alter spatial learning memory and the expression of some important metabolites that are linked to neurophysiological disorders in young rats, with the female rats showing higher susceptibility compared to the males. This indicates that female rats are more likely to show early symptoms of the disorder on exposure to chronic GBH compared to male rats. We observed that four important metabolites (paraxanthine, epinephrine, L-(+)-arginine, and D-arginine) showed significant changes and involvement in neurological changes as suggested by ingenuity pathway analysis. In conclusion, our results indicate that chronic exposure to GBH can increase the risk of developing neurological disorders.

Variations in O-Glycosylation Patterns Influence Viral Pathogenicity, Infectivity, and Transmissibility in SARS-CoV-2 Variants.

The highly glycosylated S protein plays a vital role in host cell invasion, making it the principal target for vaccine development. Differences in mutations observed on the spike (S) protein of SARS-CoV-2 variants may result in distinct glycosylation patterns, thus influencing immunological evasion, infectivity, and transmissibility. The glycans can mask key epitopes on the S1 protein and alter its structural conformation, allowing the virus to escape the immune system. Therefore, we comprehensively characterize O-glycosylation in eleven variants of SARS-CoV-2 S1 subunits to understand the differences observed in the biology of the variants. In-depth characterization was performed with a double digestion strategy and an efficient LC-MS/MS approach. We observed that O-glycosylation is highly conserved across all variants in the region between the NTD and RBD, whereas other domains and regions exhibit variation in O-glycosylation. Notably, omicron has the highest number of O-glycosylation sites on the S1 subunit. Also, omicron has the highest level of sialylation in the RBD and RBM functional motifs. Our findings may shed light on how differences in O-glycosylation impact viral pathogenicity in variants of SARS-CoV-2 and facilitate the development of a robust vaccine with high protective efficacy against the variants of concern.

N-Glycome Profile of the Spike Protein S1: Systemic and Comparative Analysis from Eleven Variants of SARS-CoV-2.

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and is therefore one of the major research targets. The S1 protein is extensively glycosylated, and there is compelling evidence that glycans protect the virus' active site from the human defense system. Therefore, investigation of the S1 protein glycome alterations in the different virus variants will provide a view of the glycan evolution and its relationship with the virus pathogenesis. In this study, we explored the N-glycosylation expression of the S1 protein for eleven SARS-CoV-2 variants: five variants of concern (VOC), including alpha, beta, gamma, delta, and omicron, and six variants of interest (VOI), including epsilon, eta, iota, lambda, kappa, and mu. The results showed significant differences in the N-glycome abundance of all variants. The N-glycome of the VOC showed a large increase in the abundance of sialofucosylated glycans, with the greatest abundance in the omicron variant. In contrast, the results showed a large abundance of fucosylated glycans for most of the VOI. Two glycan compositions, GlcNAc4,Hex5,Fuc,NeuAc (4-5-1-1) and GlcNAc6,Hex8,Fuc,NeuAc (6-8-1-1), were the most abundant structures across all variants. We believe that our data will contribute to understanding the S1 protein's structural differences between SARS-CoV-2 mutations.

Isomeric separation of native N-glycans using nano zwitterionic- hydrophilic interaction liquid chromatography column.

Changes in the expression of glycan isomers have been implicated in the development and progression of several diseases. However, the analysis of structurally diverse isomeric N-glycans by LC-MS/MS is still a major analytical challenge, particularly due to their large number of possible isomeric conformations. Common approaches derivatized the N-glycans to increase their hydrophobicity and to gain better detection in the MS system. Unfortunately, glycan derivatization is time-consuming and, in many cases, adds complexity because of the multiple reaction and cleaning steps, incomplete chemical labeling, possible degradation, and unwanted side reactions. Thus, analysis of native glycans, especially for samples with low abundance by LC-MS/MS, is desirable. Normal phase chromatography, which employs HILIC stationary phase, has been commonly employed for the identification and separation of labeled glycans. In this study, we focused on achieving efficient isomeric separation of native N-glycans using a nano ZIC-HILIC column commonly employed to separate labeled glycans and glycopeptides. Underivatized sialylated and oligomannose N-glycans derived from bovine fetuin and Ribonuclease B were initially utilized to optimize chromatographic conditions, including column temperature, pH of mobile phases, and gradient elution time. The optimized condition was then applied for the isomeric separation of native N-glycans derived from alpha-1 acid glycoprotein, as well as from biological samples. Finally, we confirmed the stability and reproducibility of the ZIC-HILIC column by performing run-to-run comparisons of the full width at half height (FWHM) and retention time on different N-glycans. The variability in FWHM was less than 0.5 min, while that of retention time was less than 1.0 min with %RSD less than 1.0%.

An Efficient and Economical N-Glycome Sample Preparation Using Acetone Precipitation.

Due to the critical role of the glycome in organisms and its close connections with various diseases, much time and effort have been dedicated to glycomics-related studies in the past decade. To achieve accurate and reliable identification and quantification of glycans extracted from biological samples, several analysis methods have been well-developed. One commonly used methodology for the sample preparation of N-glycomics usually involves enzymatic cleavage by PNGase F, followed by sample purification using C18 cartridges to remove proteins. PNGase F and C18 cartridges are very efficient both for cleaving N-glycans and for protein removal. However, this method is most suitable for a limited quantity of samples. In this study, we developed a sample preparation method focusing on N-glycome extraction and purification from large-scale biological samples using acetone precipitation. The N-glycan yield was first tested on standard glycoprotein samples, bovine fetuin and complex biological samples, and human serum. Compared to C18 cartridges, most of the sialylated N-glycans from human serum were detected with higher abundance after acetone precipitation. However, C18 showed a slightly higher efficiency for protein removal. Using the unfiltered human serum as the baseline, around 97.7% of the proteins were removed by acetone precipitation, while more than 99.9% of the proteins were removed by C18 cartridges. Lastly, the acetone precipitation was applied to N-glycome extraction from egg yolks to demonstrate large-scale glycomics sample preparation.