Construction of an InstantPC-derivatized glycan glucose unit database: A foundation work for high-throughput and high-sensitivity glycomic analysis.

The glycosylation profile of biotherapeutic glycoproteins is a critical quality attribute that is routinely monitored to ensure desired product quality, safety and efficacy. Additionally, as one of the most prominent and complex post-translational modifications, glycosylation plays a key role in disease manifestation. Changes in glycosylation may serve as a specific and sensitive biomarker for disease diagnostics and prognostics. However, the conventional 2-aminobenzamide-based N-glycosylation analysis procedure is time-consuming and insensitive with poor reproducibility. We have evaluated an innovative streamlined 96-well-plate-based platform utilizing InstantPC label for high-throughput, high-sensitivity glycan profiling, which is user-friendly, robust and ready for automation. However, the limited availability of InstantPC-labeled glycan standards has significantly hampered the applicability and transferability of this platform for expedited glycan structural profiling. To address this challenge, we have constructed a detailed InstantPC-labeled glycan glucose unit (GU) database through analysis of human serum and a variety of other glycoproteins from various sources. Following preliminary hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection separation and analysis, glycoproteins with complex glycan profiles were subjected to further fractionation by weak anion exchange HILIC and exoglycosidase sequential digestion for cross-validation of the glycan assignment. Hydrophilic interaction ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry was subsequently utilized for glycan fragmentation and accurate glycan mass confirmation. The constructed InstantPC glycan GU database is accurate and robust. It is believed that this database will enhance the application of the developed platform for high-throughput, high-sensitivity glycan profiling and that it will eventually advance glycan-based biopharmaceutical production and disease biomarker discovery.

Immobilized peptide-N-glycosidase F onto magnetic nanoparticles: A biotechnological tool for protein deglycosylation under native conditions.

The elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%-80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.

A nomogram based on glycomic biomarkers in serum and clinicopathological characteristics for evaluating the risk of peritoneal metastasis in gastric cancer.

BACKGROUND: Peritoneal metastasis (PM) in gastric cancer (GC) remains an untreatable disease, and is difficult to diagnose preoperatively. Here, we aim to establish a novel prediction model. METHODS: The clinicopathologic characteristics of a cohort that included 86 non-metastatic GC patients and 43 PMGC patients from Zhongshan Hospital were retrospectively analysed to identify PM associated variables. Additionally, mass spectrometry and glycomic analysis were applied in the same cohort to find glycomic biomarkers in serum for the diagnosis of PM. A nomogram was established based on the associations between potential risk variables and PM. RESULTS: Overexpression of 4 N-glycans (H6N5L1E1: m/z 2620.93; H5N5F1E2: m/z 2650.98; H6N5E2, m/z 2666.96; H6N5L1E2, m/z 2940.08); weight loss ≥ 5 kg; tumour size ≥ 3 cm; signet ring cell or mucinous adenocarcinoma histology type; poor differentiation; diffuse or mixed Lauren classification; increased CA19-9, CA125, and CA724 levels; decreased lymphocyte count, haemoglobin, albumin, and pre-albumin levels were identified to be associated with PM. A nomogram that integrated with five independent risk factors (weight loss ≥ 5 kg, CA19-9 ≥ 37 U/mL, CA125 ≥ 35 U/mL, lymphocyte count < 2.0 * 10 ~ 9/L, and H5N5F1E2 expression ≥ 0.0017) achieved a good performance for diagnosis (AUC: 0.892, 95% CI 0.829-0.954). When 160 was set as the cut-off threshold value, the proposed nomogram represented a perfectly discriminating power for both sensitivity (0.97) and specificity (0.88). CONCLUSIONS: The nomogram achieved an individualized assessment of the risk of PM in GC patients; thus, the nomogram could be used to assist clinical decision-making before surgery.

Diagnostic Challenges of Ocular Rosacea.

OBJECTIVE: This study aims to address the issues surrounding the diagnosis of ocular rosacea and to evaluate the development of the patients' condition after treatment, as well as to distinguish between healthy and diseased patients using a glycomic analysis of tears. METHODOLOGY: A prospective study was conducted to assess a total of 68 eyes in 34 patients over a six-week period. These patients were diagnosed with ocular rosacea based on subjective symptoms and clinical examination. The study monitored the development of objective and subjective values. The difference between patients with the pathology and healthy controls was established by means of analysis of glycans in tears. RESULTS: Skin lesions were diagnosed in 94% of patients with ocular rosacea, with the most commonly observed phenotype being erythematotelangiectatic (68.8%). The mean duration of symptoms was 29.3 months (range 0.5­126 months) with a median of 12 months. Throughout the study, an improvement in all monitored parameters was observed, including Meibomian gland dysfunction, bulbar conjunctival hyperemia, telangiectasia of the eyelid margin, anterior blepharitis, uneven and reddened eyelid margins, and corneal neovascularization. The study also observed improvements in subjective manifestations of the disease, such as foreign body sensation, burning, dryness, lachrymation, itching eyes, photophobia, and morning discomfort. The analysis of glycans in tears partially separated tear samples based on their origin, which allowed for the differentiation of patients with rosacea from healthy controls. In the first sample, the pathology was determined in a total of 63 eyes (98.4%) of 32 patients, with further samples showing a change in the glycomic profile of patients' tears during treatment. CONCLUSION: The study demonstrated objective and subjective improvements in all the patients. Tear sampling and analysis could provide a means of timely diagnosis of ocular rosacea.

Effects of Blanching, Freezing and Canning on the Carbohydrates in Sweet Corn.

Sweet corn is frequently consumed in the US and contains carbohydrates as major macronutrients. This study examined the effects of blanching, freezing, and canning on carbohydrates in sweet corn. Fresh bi-color sweet corn was picked in the field and processed immediately into frozen and canned samples. Simple sugars, starch, and dietary fiber (DF) (including total DF (TDF), insoluble DF (IDF) and two fractions of soluble DF (SDF)) were measured according to the AOAC methods. Additional glycomic analysis including oligosaccharides, monosaccharide composition of total polysaccharides (MCTP) and glycosidic linkage of total polysaccharides (GLTP) were analyzed using UHPLC-MS. Sucrose is the major simple sugar, and IDF is the main contributor to TDF. Sucrose and total simple sugar concentrations were not altered after blanching or freezing but were significantly reduced in canned samples. Kestose was the only oligosaccharide identified in sweet corn and decreased in all heat-treated or frozen samples. Starch content decreased in frozen samples but increased in canned samples. While two SDF fractions did not differ across all samples, blanching, freezing and canning resulted in increases in TDF and IDF. Six monosaccharides were identified as major building blocks of the total polysaccharides from MCTP analysis. Glucose and total monosaccharide concentrations increased in two canned samples. GLTP was also profoundly altered by different food processing methods. This study provided insights into the changes in the content and quality of carbohydrates in sweet corn after food processing. The data are important for accurate assessment of the carbohydrate intake from different sweet corn products.

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens.

BACKGROUND: Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation. METHODS: Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry. RESULTS: Clostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles. CONCLUSIONS: Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes. GENERAL SIGNIFICANCE: Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.

Immobilization of ß-galactosidase and α-mannosidase onto magnetic nanoparticles: A strategy for increasing the potentiality of valuable glycomic tools for glycosylation analysis and biological role determination of glycoconjugates.

Glycans present in biological glycoconjugates have several structural and functional roles. Elucidation of glycan structure and biological function is critical to understand their role in physiological and pathogenic process, enabling the development of diagnostic methods and disease treatment. Immobilized glycosidases are powerful tools for glycan analysis, as they are able to remove specific carbohydrates without altering the protein structure. Here we describe the individual immobilization of Aspergillus oryzae ß-galactosidase and Canavalia ensiformis α-mannosidase onto agarose and silica magnetic nanoparticles activated with cyanate ester groups. High immobilization yields (70-90%) were achieved, keeping above 60% of its original activity. Immobilized glycosidases were effective in the selective deglycosylation of model glycoproteins and a Fasciola hepatica lysate, evidenced by a decrease in specific lectin recognition of 40-50% after enzymatic deglycosylation. Immobilized glycosidases were reused for several deglycosylation cycles without loss of effectiveness. Their use was extended to the elucidation of the glycan role of native glycoconjugates. A decrease in the recognition of lactoferrin treated with α-mannosidase by a C-type lectin receptor, DC-SIGN was found. Also the specific deglycosylation of a F. hepatica lysate demonstrated the relevance of mannosylated glycans in the induction of Th2/Treg immune responses during the infection. Our results show successful immobilization of specific glycosidases in nano-supports and validate their utility to identify glycans biological functions.

Discovery of Non-invasive Glycan Biomarkers for Detection and Surveillance of Gastric Cancer.

Purpose: Gastric cancer (GC), one of the world's top five most common cancers, is the third leading cause of cancer related death. It is urgent to identify non-invasive biomarkers for GC. The objective of our study was to find out non-invasive biomarkers for early detection and surveillance of GC based on glycomic analysis. Method: Ethyl esterification derivatization combined with MALDI-TOF MS analysis was employed for the comprehensive serum glycomic analysis in order to investigate glycan markers that would indicate the onset and progression of gastric cancer. Upon the discovery of the candidate biomarkers, those with great potential were further validated in an independent test set. Peaks were acquired by the software of MALDI-MS sample acquisition and processing and analyzed by the software of Progenesis MALDI. Results: The differences in glycosylation were found between non-cancer controls and gastric cancer samples: hybrid and multi-branched type (tri-, tetra-antennnary glycans) N-glycans were increased in GC, yet monoantennary, galactose, bisecting type and core fucose N-glycans were decreased. In training set, core fucose (AUC=0.923, 95%CI: 0.8485 to 0.9967) played an excellent diagnostic performance for the early detection of gastric cancer. The diagnostic potential of core fucose was further validated in an independent cohort (AUC=0.854, 95%CI: 0.7592 to 0.9483). Besides, several individual glycan structures reached both statistical criteria (p-values less than 0.05 and AUC scores that were at least moderately accurate) when comparing different stages of GC samples. Conclusion: We comprehensively evaluate the serum glycan changes in different stages of GC patients including peritoneal metastasis for the first time. We determined several N-glycan biomarkers, some of these have potential in distinguishing the early stage GC from healthy controls, and the others can help to monitor the progression of GC. The findings also enhance understanding of gastric cancer.

Solid-phase reductive amination for glycomic analysis.

Reductive amination is an indispensable method for glycomic analysis, as it tremendously facilitates glycan characterization and quantification by coupling functional tags at the reducing ends of glycans. However, traditional in-solution derivatization based approach for the preparation of reductively aminated glycans is quite tedious and time-consuming. Here, a simpler and more efficient strategy termed solid-phase reductive amination was investigated. The general concept underlying this new approach is to streamline glycan extraction, derivatization, and purification on non-porous graphitized carbon sorbents. Neutral and sialylated standard glycans were utilized to test the feasibility of the solid-phase method. As results, almost complete labeling of those glycans with four common labels of aniline, 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA) and 2-amino-N-(2-aminoethyl)-benzamide (AEAB) was obtained, and negligible desialylation occurred during sample preparation. The labeled glycans derived from glycoproteins showed excellent reproducibility in high performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Direct comparisons based on fluorescent absorbance and relative quantification using isotopic labeling demonstrated that the solid-phase strategy enabled 20-30% increase in sample recovery. In short, the solid-phase strategy is simple, reproducible, efficient, and sensitive for glycan analysis. This method was also successfully applied for N-glycan profiling of HEK 293 cells with MALDI-TOF MS, showing its attractive application in the high-throughput analysis of mammalian glycome.