Excessive Substitution of Fish Meal with Fermented Soybean Meal Induces Oxidative Stress by Impairing Glutathione Metabolism in Largemouth Bass (Micropterus salmoides).

The application of fermented soybean meal (FSBM) is an effective strategy to alleviate the shortage of fish meal (FM) in aquaculture. However, an excessive substitution ratio often reduces fish growth and induces liver oxidative stress, while the mechanism remains poorly understood. Here, an 8-week feeding trial was conducted in largemouth bass (initial weight: 6.82 ± 0.09 g) to establish an oxidative stress model by replacing 50% of FM with FSBM (fermented by Bacillus subtilis). The results showed that FSBM substitution significantly reduced the growth performance of largemouth bass, including the weight gain rate and specific growth rate. Moreover, FSBM significantly reduced the contents of essential amino acids and total free amino acids in muscle, along with the mRNA expression of amino acids and small peptide transporters. Enzyme activity detection and liver sections showed that FSBM substitution caused liver oxidative stress, indicating the successful construction of an oxidative stress model. An integrated analysis of transcriptomic and metabolomic data revealed that FSBM substitution impaired glycine, serine and threonine metabolism, as well as glutathione metabolism. In addition, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was decreased in the FSBM group, which may explain the mechanism of oxidative stress caused by FSBM substitution. Considering that glycine is an important component of glutathione synthesis, key genes involved in glycine metabolism (glya, gnmt and agxt) and dietary glycine supplementation should be valued to improve the availability of FSBM. This study reveals for the first time the importance of non-essential amino acids in improving the utilization of plant-based protein sources and provides original insight for the optimization of aquatic feeds.

SLC3A2 promotes tumor-associated macrophage polarization through metabolic reprogramming in lung cancer.

Tumor-associated macrophages (TAMs) are one of the most abundant immunosuppressive cells in the tumor microenvironment and possess crucial functions in facilitating tumor progression. Emerging evidence indicates that altered metabolic properties in cancer cells support the tumorigenic functions of TAMs. However, the mechanisms and mediators the underly the cross-talk between cancer cells and TAMs remain largely unknown. In the present study, we revealed that high solute carrier family 3 member 2 (SLC3A2) expression in lung cancer patients was associated with TAMs and poor prognosis. Knockdown of SLC3A2 in lung adenocarcinoma cells impaired M2 polarization of macrophages in a coculture system. Using metabolome analysis, we identified that SLC3A2 knockdown altered the metabolism of lung cancer cells and changed multiple metabolites, including arachidonic acid, in the tumor microenvironment. More importantly, we showed that arachidonic acid was responsible for SLC3A2-mediated macrophage polarization in the tumor microenvironment to differentiate into M2 type both in vitro and in vivo. Our data illustrate previously undescribed mechanisms responsible for TAM polarization and suggest that SLC3A2 acts as a metabolic switch on lung adenocarcinoma cells to induce macrophage phenotypic reprogramming through arachidonic acid.

Effects of Synthetic Astaxanthin on the Growth Performance, Pigmentation, Antioxidant Capacity, and Immune Response in Black Tiger Prawn (Penaeus monodon).

Synthetic astaxanthin is an effective nutritional strategy for improving shrimp body color and promoting growth. However, the optimal amount of astaxanthin in feed also varies with the synthetic technology and purity. In the present study, five diets containing different doses of synthetic astaxanthin (0% (CON), 0.02% (AX0.02), 0.04% (AX0.04), 0.08% (AX0.08), and 0.16% (AX0.16)) were administered to Penaeus monodon (initial body weight: 0.3 ± 0.03 g) for 8 weeks. With an increase in astaxanthin content in feed, weight gain and specific growth rate increased initially and subsequently decreased, with the highest value appearing at AX0.08. Dietary astaxanthin supplementation obviously improved the carapace and muscle color by enhancing astaxanthin pigmentation. Meanwhile, the fatty acid profile was altered by dietary astaxanthin, as evidenced by a decline in palmitic acid proportion, along with an increase in n-3 polyunsaturated fatty acids (n-3 PUFA) contents in muscle. In addition, dietary astaxanthin supplementation regulated prawn's antioxidant capacity. In the hemolymph, the activities of glutamic pyruvic transaminase (GPT) showed a significantly decrease trend with linear effect. The activities of glutamic oxaloacetic transaminase (GOT) and the contents of malondialdehyde (MDA) were first downregulated and then upregulated with significantly quadratic pattern. In the hepatopancreas, the activities of superoxide dismutase (SOD) and the contents of MDA were significantly downregulated with the increase of dietary astaxanthin levels. Reduced glutathione (GSH) contents and catalase (CAT) activities were also significantly decreased in group AX0.08. Correspondingly, astaxanthin decreased GSH and MDA contents under transportation stress. Moreover, the mRNA expression of immune genes (traf6, relish, and myd88) were inhibited by dietary astaxanthin supplementation. Based on the results of polynomial contrasts analysis and Duncan's test, dietary synthetic astaxanthin is a suitable feed additive to improve the growth, body color, antioxidant capacity, and nonspecific immunity of P. monodon. According to the second-order polynomial regression analysis based on the weight gain, the optimal supplementation level of dietary astaxanthin was 90 mg kg-1 in P. monodon.

Girdin Promotes Tumorigenesis and Chemoresistance in Lung Adenocarcinoma by Interacting with PKM2.

Girdin, an Akt substrate, has been reported to promote tumorigenesis in various tumors. However, the role of Girdin in a spontaneous tumor model has not yet been explored. Here, we studied the role of Girdin in lung adenocarcinoma (LUAD) using the autochthonous mouse model and found that Girdin led to LUAD progression and chemoresistance by enhancing the Warburg effect. Mechanistically, Girdin interacted with pyruvate kinase M2 (PKM2), which played a vital role in aerobic glycolysis. Furthermore, Girdin impaired Platelet Derived Growth Factor Receptor Beta (PDGFRß) degradation, which in turn, promoted PKM2 tyrosine residue 105 (Y105) phosphorylation and inhibited PKM2 activity, subsequently promoting aerobic glycolysis in cancer cells. Taken together, our study demonstrates that Girdin is a crucial regulator of tumor growth and may be a potential therapeutic target for overcoming the resistance of LUAD cells to chemotherapy.

A Novel Integrated Metabolism-Immunity Gene Expression Model Predicts the Prognosis of Lung Adenocarcinoma Patients.

Background: Although multiple metabolic pathways are involved in the initiation, progression, and therapy of lung adenocarcinoma (LUAD), the tumor microenvironment (TME) for immune cell infiltration that is regulated by metabolic enzymes has not yet been characterized. Methods: 517 LUAD samples and 59 non-tumor samples were obtained from The Cancer Genome Atlas (TCGA) database as the training cohort. Kaplan-Meier analysis and Univariate Cox analysis were applied to screen the candidate metabolic enzymes for their role in relation to survival rate in LUAD patients. A prognostic metabolic enzyme signature, termed the metabolic gene risk score (MGRS), was established based on multivariate Cox proportional hazards regression analysis and was verified in an independent test cohort, GSE31210. In addition, we analyzed the immune cell infiltration characteristics in patients grouped by their Risk Score. Furthermore, the prognostic value of these four enzymes was verified in another independent cohort by immunohistochemistry and an optimized model of the metabolic-immune protein risk score (MIPRS) was constructed. Results: The MGRS model comprising 4 genes (TYMS, NME4, LDHA, and SMOX) was developed to classify patients into high-risk and low-risk groups. Patients with a high-risk score had a poor prognosis and exhibited activated carbon and nucleotide metabolism, both of which were associated with changes to TME immune cell infiltration characteristics. In addition, the optimized MIPRS model showed more accurate predictive power in prognosis of LUAD. Conclusion: Our study revealed an integrated metabolic enzyme signature as a reliable prognostic tool to accurately predict the prognosis of LUAD.

Suppression of DLBCL Progression by the E3 Ligase Trim35 Is Mediated by CLOCK Degradation and NK Cell Infiltration.

The majority of diffuse large B-cell lymphoma (DLBCL) patients develop relapsed or refractory disease after standard ruxolitinib, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemotherapy, which is partly related to a dysregulated tumor immune microenvironment. However, how the infiltration of immune cells is appropriately regulated is poorly understood. Herein, we show that the E3 ubiquitin ligase Trim35 is expressed at low levels in human DLBCL tissues. We also show that overexpression of Trim35 suppresses DLBCL cell proliferation and correlates with inferior survival in DLBCL patients. Our mechanistic study shows that Trim35 functions as an E3 ligase to mediate the ubiquitination and degradation of CLOCK, a key regulator of circadian rhythmicity. High expression of Trim35 correlates with NK cell infiltration in DLBCL, partly due to the degradation of CLOCK. Consistently, patients with high expression of CLOCK show poor overall survival. Overall, these findings suggest that Trim35 suppresses the progression of DLBCL by modulating the tumor immune microenvironment, indicating that it may be a promising diagnostic and prognostic biomarker in DLBCL.

Diagnostic Value of Serum Concentration of Galectin-3 in Patients With Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Although the predictive value of galectin-3 for heart failure with preserved ejection fraction has been demonstrated, the diagnostic value remains unclear. The present study was performed to address this issue. HYPOTHESIS: Galectin-3 has diagnostic value for heart failure with preserved ejection fraction. METHODS: This is a diagnostic experiment. We conducted an observational study of 223 patients with combined symptoms of heart failure and diseases that can lead to heart failure with preserved ejection fraction. Patients were grouped into the heart failure group and control group in accordance with the 2016 European Society of Cardiology heart failure guidelines for heart failure with preserved ejection fraction. Baseline information and serum galectin-3 concentration were assessed within 24 h after admission. RESULTS: Serum galectin-3 concentration was significantly higher in the heart failure group compared with the control group. Binary logistic regression analysis showed that higher galectin-3 concentration was associated with the occurrence of heart failure with preserved ejection fraction. The area under the curve of galectin-3 was 0.763, indicating that galectin-3 has moderate diagnostic value for heart failure with preserved ejection fraction. Galectin-3 >15.974 ng/mL identified heart failure with preserved ejection fraction with 76.0% sensitivity and 71.9% specificity. CONCLUSIONS: There was a correlation between galectin-3 and heart failure with preserved ejection fraction, and galectin-3 was an independent predictor of heart failure with preserved ejection fraction. The diagnostic value of galectin-3 for heart failure with preserved ejection fraction was moderate (AUC: 0.763, 95% CI: 0.696-0.821, P < 0.01, and the sensitivity is 76.0% while the specificity is 71.9% at the threshold 15.974 ng/mL) and was higher than that of interventricular septal thickness or E/A ratio.

Heat map visualization for electrocardiogram data analysis.

BACKGROUND: Most electrocardiogram (ECG) studies still take advantage of traditional statistical functions, and the results are mostly presented in tables, histograms, and curves. Few papers display ECG data by visual means. The aim of this study was to analyze and show data for electrocardiographic left ventricular hypertrophy (LVH) with ST-segment elevation (STE) by a heat map in order to explore the feasibility and clinical value of heat mapping for ECG data visualization. METHODS: We sequentially collected the electrocardiograms of inpatients in the First Affiliated Hospital of Shantou University Medical College from July 2015 to December 2015 in order to screen cases of LVH with STE. HemI 1.0 software was used to draw heat maps to display the STE of each lead of each collected ECG. Cluster analysis was carried out based on the heat map and the results were drawn as tree maps (pedigree maps) in the heat map. RESULTS: In total, 60 cases of electrocardiographic LVH with STE were screened and analyzed. STE leads were mainly in the V1, V2 and V3 leads. The ST-segment shifts of each lead of each collected ECG could be conveniently visualized in the heat map. According to cluster analysis in the heat map, STE leads were clustered into two categories, comprising of the right precordial leads (V1, V2, V3) and others (V4, V5, V6, I, II, III, aVF, aVL, aVR). Moreover, the STE amplitude in 40% (24 out of 60) of cases reached the threshold specified in the STEMI guideline. These cases also could be fully displayed and visualized in the heat map. Cluster analysis in the heat map showed that the III, aVF and aVR leads could be clustered together, the V1, V2, V3 and V4 leads could be clustered together, and the V5, V6, I and aVL leads could be clustered together. CONCLUSION: Heat maps and cluster analysis can be used to fully display every lead of each electrocardiogram and provide relatively comprehensive information.

[Targeting EGFRvIII for treatment of glioblastoma: From molecular mechanisms to clinical strategies].

Glioblastoma is one of the most common intracranial malignant tumor and its initiation and progression are closely associated with epidermal growth factor receptor (EGFR). EGFR variant III (EGFRvIII) is a mutant EGFR and highly expressed in glioblastoma. EGFRvIII promotes the proliferation and invasiveness of glioblastoma cells and induces drug resistance by signaling networks.

Elevated survivin expression in peripheral blood mononuclear cells is central to collateral formation in coronary chronic total occlusion.

Survivin is essential to angiogenesis and revascularization, but its role in coronary collateral formation remains unclear. The role of survivin in peripheral blood mononuclear cells (PBMCs) of coronary chronic total occlusion (CTO) patients was investigated. Coronary CTO patients (n=46; mean age 60.1±8.5, male 54.3%) (CTO group) and normal control patients (n=18; mean age 58.0±10.0, male 55.6%) underwent angiographic collateral vessel grading by Rentrop classification (C0 - C3) and provided peripheral blood between June 2006 and February 2007. Rat hind limb ischemia models were constructed using four equal groups of Sprague-Dawley rats (n=36): normal control, sham operation, operation and granulocyte macrophage colony-stimulating factor (GM-CSF). PBMC numbers and characteristics, collateral vessels, survivin, CD4, CD8, CD44, vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) expression were determined using RT-PCR, flow cytometry, immunocytochemistry and western blot analysis. PBMC survivin mRNA and protein expression levels were higher in patients with good collateral circulation (C2 + C3) than in patients with no collateral flow (C0) (all P<0.05). Survivin single-positive and survivin and CD8, VEGF and ICAM-1 double-positive percentages were elevated in patients with good collateral circulation compared to those with normal and no collateral flow (all P<0.05), consistent with the rat model results, wherein higher survivin levels produced significantly larger and more visible collateral vessels. In conclusion, elevated survivin expression in PBMCs, particularly survivin and CD8, VEGF, and ICAM-1 double-positive PBMCs, may be crucial for good collateral formation in patients with coronary CTO, as confirmed by assessment of a rat model.

Germanium in ginseng is low and causes no sodium and water retention or renal toxicity in the diuretic-resistant rats.

Ginseng preparations contain high concentrations of germanium (Ge), which was reported to contribute to diuretic resistance or renal failure. However, Ge content in ginseng and the influence on renal functions remain unclear. Forty rats were randomly divided into control group, low, moderate, and high Ge ginseng-treated group and observed for 25 days. Daily urine, renal functions, and serum and urine electrolytics were measured. Ge retention in the organs and renal histological changes were also evaluated. Ge content ranged from 0.007 to 0.450 µg/g in various ginseng samples. Four groups showed no difference in the daily urine output, glomerular filtration rate, urinary electrolytes excretions, 24 h-urine protein, as well as plasma and urine urea nitrogen, creatinine, osmotic pressure, and pH values. Ge did not cause any renal pathological effects in this study. No Na and water retention was detected in the ginseng-treated groups. Ge retention in various organs was found highest in spleen, followed by the kidney, liver, lung, stomach, heart, and pancreas. The total Ge contents in various ginsengs were low, and ginseng treatment did not affect renal functions or cause renal histological changes.

Serum ubiquitin via CXC chemokine receptor 4 triggered cyclooxygenase-1 ubiquitination possibly involved in the pathogenesis of aspirin resistance.

Extracellular ubiquitin (Ub) with platelet aggregation property was found higher in acute myocardial infarction (AMI) patients. Here we detected the platelet functions and serum Ub levels in 250 AMI patients and 50 healthy volunteers before and after aspirin treatment. The influence of serum Ub on platelet functions was determined in vitro. We found that 47 out of 250 AMI patients showed aspirin resistance (AR) and 203 showed aspirin sensitivity (AS). During hospitalization, AR group had higher serum Ub levels than the AS group or the healthy group, and the serum Ub levels was related to the rates of thrombosis events. The patients with higher serum Ub levels showed that the platelets had more ubiquitinated platelets, higher contents of ubiquitinated proteins and ubiquitinated cyclooxygenase-1 (COX-1). The levels of ubiquitinated COX-1 in the platelets was inversely correlated with acetylated COX-1, the separated ubiquitinated COX-1 activity was approximately twofold or fourfold higher than the total COX-1(ubiquitinated COX-1 and COX-1) or COX-1. In vitro, we found that extracellular Ub, via the CXC chemokine receptor 4 (CXCR4) pathway, facilitated COX-1 to be ubiquitined and prevented aspirin to acetylate its target. Platelets had higher levels of ubiquitinated COX-1 showing poor response to aspirin. Such results were not detected in Ub-free serum or ovalbumin incubated platelets. Serum Ub, via the CXCR4 pathway, facilitated COX-1 to be ubiquitined and activated the platelets possibly involved in the pathogenesis of AR.

Hypoxia-inducible factor-1α polymorphisms link to coronary artery collateral development and clinical presentation of coronary artery disease.

AIMS: This study aimed to investigate the association of Hypoxia-inducible factor-1α (HIF-1α) C1772T and G1790A single nucleotide polymorphisms (SNPs) with: incidence, clinical type, severity of coronary atherosclerosis and coronary collaterals of coronary artery disease (CAD). METHODS: The clinical data and genomic DNA were gathered in 958 subjects, including 560 controls and 398 patients with CAD. CAD was confirmed with coronary angiography (CAG). The genotypes for two SNPs were determined by high resolution melting after PCR amplification. RESULTS: Neither the HIF-1α C1772T nor the G1790A genotype was significantly associated with CAD and, no gene-gene or gene-environmental interactions were identified. However, both HIF-1α C1772T and G1790A (P<0.05) alleles were associated with clinical type and formation of coronary collaterals (P<0.05). Patients carrying genotype CT (P=0.019, OR=4.905,91, 95% CI: 1.355-17.761) and GA (P=0.026, OR=3.052, 95% CI: 1.180-7.892) had significantly higher stable angina pectoris (SAP) than unstable angina pectoris (UAP) and acute myocardial infarction (AMI). The presence of HIF-1 genotype CT (P=0.016, OR=13.373, 95% CI: 15.468-32.709) and GA (P=0.001, OR=19.741, 95% CI: 8.125-47.966) predicted lower collateral formation and severity of CAD secondary to the absence of collaterals (r=0.242, P<0.001). CONCLUSIONS: We conclude that functional polymorphisms in the HIF-1α gene do not modify CAD risk but they are associated with the formation of coronary collaterals and clinical presentation of CAD.

Autoantibody biomarkers identified by proteomics methods distinguish ovarian cancer from non-ovarian cancer with various CA-125 levels.

PURPOSE: CA-125 has been a valuable marker for detecting ovarian cancer, however, it is not sensitive enough to detect early-stage disease and not specific to ovarian cancer. The purpose of our study was to identify autoantibody markers that are specific to ovarian cancer regardless of CA-125 levels. METHODS: Top-down and iTRAQ quantitative proteomics methods were used to identify high-frequency autoantibodies in ovarian cancer. Protein microarrays comprising the recombinant autoantigens were screened using serum samples from various stages of ovarian cancer with diverse levels of CA-125 as well as benign and healthy controls. ROC curve and dot blot analyses were performed to validate the sensitivity and specificity of the autoantibody markers. RESULTS: The proteomics methodologies identified more than 60 potential high-frequency autoantibodies in ovarian cancer. Individual serum samples from ovarian cancer stages I-IV compared to control samples that were screened on a microarray containing native recombinant autoantigens revealed a panel of stage I high-frequency autoantibodies. Preliminary ROC curve and dot blot analyses performed with the ovarian cancer samples showed higher specificity and sensitivity as compared to CA-125. Three autoantibody markers exhibited higher specificity in various stages of ovarian cancer with low and normal CA-125 levels. CONCLUSIONS: Proteomics technologies are suitable for the identification of protein biomarkers and also the identification of autoantibody biomarkers when combined with protein microarray screening. Using native recombinant autoantigen arrays to screen autoantibody markers, it is possible to identify markers with higher sensitivity and specificity than CA-125 that are relevant to early detection of ovarian cancer.

Chronic aspirin via dose-dependent and selective inhibition of cardiac proteasome possibly contributed a potential risk to the ischemic heart.

Impaired cardiac proteasome has been reported in ischemic heart and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, it's unclear whether it affects cardiac proteasome functions. Myocardial infarction (MI), sham or normal male SD rats were injected intraperitoneally with high (300 mg/kg), low (5 mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated human ventricular myocytes. Dose-related increases in heart and ventricular weight, and impaired cardiac functions, were found more exacerbated in the aspirin-treated MI rat hearts than the saline-treated MI counterparts. The activity of 26S, 20S and 19S declined by about 30%, or the 20S proteasome subunits ß5, ß2 and ß1 decreased by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats (P<0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, ß5 by 30% and 12%, and ß1 by 40% and 30%, respectively, and the lost activity was correlated with the compromised cardiac functions or the decreased cell viability. The dose-related and selective inhibition of 26S and 20S proteasome, or the 20S proteasome subunits ß5 and ß1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. The impaired cardiac proteasome, enhanced by chronic aspirin treatment, attenuated the removal of oxidative and ubiquitinated proteins, and chronic aspirin treatment via selective and dose-dependent inhibition of cardiac proteasome possibly constituted a potential risk to ischemic heart.

Chinese medicine for mental disorder and its applications in psychosomatic diseases.

With the development of modern medicine, an increasing awareness has developed regarding the limitations of a specialized and compartmentalized approach to clinical practice that largely ignores the interconnectedness of the mind, body, and spirit. Although contemporary medicine now accepts this interconnectedness, practitioners tend to think that the emotions play a secondary or excitatory role in producing disease rather than being a primary causative factor. Traditional Chinese medicine (TCM), which stems from Confucianism, Buddhism, and Daoism, views the body and the spirit as inseparable. This construct provides the foundation for the whole system of TCM, and therefore constitutes the backbone of TCM. This article presents the ways in which emotion can act as an internal etiological factor that produces a pathogenic mechanism and that underlies various psychosomatic diseases. Therefore, this article intends to integrate the ancient classic treatise established in the Yellow Emperor's Canon of Internal Medicine with current data. Likewise, the authors discuss their empirical experience to illustrate the following concepts: (1) the factors contributing to emotional impairment; (2) the holistic approach to diagnosing psychosomatic disease; (3) the integrative therapy necessary to restore the balance of body and mind; and (4) the role of emotional theory in nursing care and the prevention of psychosomatic disease.

Aleuria Aurantia Lectin (AAL)-reactive immunoglobulin G rapidly appears in sera of animals following antigen exposure.

We have discovered an Aleuria Aurantia Lectin (AAL)-reactive immunoglobulin G (IgG) that naturally occurs in the circulation of rabbits and mice, following immune responses induced by various foreign antigens. AAL can specifically bind to fucose moieties on glycoproteins. However, most serum IgGs are poorly bound by AAL unless they are denatured or treated with glycosidase. In this study, using an immunogen-independent AAL-antibody microarray assay that we developed, we detected AAL-reactive IgG in the sera of all animals that had been immunized 1-2 weeks previously with various immunogens with and without adjuvants and developed immunogen-specific responses. All of these animals subsequently developed immunogen-specific immune responses. The kinetics of the production of AAL-reactive IgG in mice and rabbits were distinct from those of the immunogen-specific IgGs elicited in the same animals: they rose and fell within one to two weeks, and peaked between four to seven days after exposure, while immunogen-specific IgGs continued to rise during the same period. Mass spectrometric profiling of the Fc glycoforms of purified AAL-reactive IgGs indicates that these are mainly comprised of IgGs with core-fucosylated and either mono-or non-galactosylated Fc N-glycan structures. Our results suggest that AAL-reactive IgG could be a previously unrecognized IgG subset that is selectively produced at the onset of a humoral response.

Chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples.

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.