α2,3-Sialylation with Fucosylation Associated with More Severe Anti-MDA5 Positive Dermatomyositis Induced by Rapidly Progressive Interstitial Lung Disease.

Dermatomyositis (DM) is a heterogeneous autoimmune disease associated with numerous myositis specific antibodies (MSAs) in which DM with anti-melanoma differentiation-associated gene 5-positive (MDA5 + DM) is a unique subtype of DM with higher risk of developing varying degrees of Interstitial lung disease (ILD). Glycosylation is a complex posttranslational modification of proteins associated with many autoimmune diseases. However, the association of total plasma N-glycome (TPNG) and DM, especially MDA5 + DM, is still unknown. TPNG of 94 DM patients and 168 controls were analyzed by mass spectrometry with in-house reliable quantitative method called Bionic Glycome method. Logistic regression with age and sex adjusted was used to reveal the aberrant glycosylation of DM and the association of TPNG and MDA5 + DM with or without rapidly progressive ILD (RPILD). The elastic net model was used to evaluate performance of glycans in distinguishing RPLID from non-RPILD, and survival analysis was analyzed with N-glycoslyation score by Kaplan-Meier survival analysis. It was found that the plasma protein N-glycome in DM showed higher fucosylation and bisection, lower sialylation (α2,3- not α2,6-linked) and galactosylation than controls. In MDA5 + DM, more severe disease condition was associated with decreased sialylation (specifically α2,3-sialylation with fucosylation) while accompanying elevated H6N5S3 and H5N4FSx, decreased galactosylation and increased fucosylation and the complexity of N-glycans. Moreover, glycosylation traits have better discrimination ability to distinguish RPILD from non-RPILD with AUC 0.922 than clinical features and is MDA5-independent. Survival advantage accrued to MDA5 + DM with lower N-glycosylation score (p = 3e-04). Our study reveals the aberrant glycosylation of DM for the first time and indicated that glycosylation is associated with disease severity caused by ILD in MDA5 + DM, which might be considered as the potential biomarker for early diagnosis of RPILD and survival evaluation of MDA5 + DM. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00096-z.

Generalized low levels of serum N-glycans associate with better health status.

Caloric restriction (CR) can prolong life and ameliorate age-related diseases; thus, its molecular basis might provide new insights for finding biomarker and intervention for aging and age-related disease. Glycosylation is an important post-translational modification, which can timely reflect the changes of intracellular state. Serum N-glycosylation was found changed with aging in humans and mice. CR is widely accepted as an effective anti-aging intervention in mice and could affect mouse serum fucosylated N-glycans. However, the effect of CR on the level of global N-glycans remains unknown. In order to explore whether CR affect the level of global N-glycans, we performed a comprehensive serum glycome profiling in mice of 30% calorie restriction group and ad libitum group at 7 time points across 60 weeks by MALDI-TOF-MS. At each time point, the majority of glycans, including galactosylated and high mannose glycans, showed a consistent low level in CR group. Interestingly, O-acetylated sialoglycans presented an upward change different from other derived traits, which is mainly reflected in two biantennary α2,6-linked sialoglycans (H5N4Ge2Ac1, H5N4Ge2Ac2). Liver transcriptome analysis further revealed a decreased transcriptional level of genes involved in N-glycan biosynthesis while increased level of acetyl-CoA production. This finding is consistent with changes in serum N-glycans and O-acetylated sialic acids. Therefore, we provided one possible molecular basis for the beneficial effect of CR from N-glycosylation perspective.

Serum IgG N-glycans enable early detection and early relapse prediction of colorectal cancer.

Colorectal cancer (CRC) develops mainly from colorectal advanced adenomas (AA), which are considered precancerous lesions. Novel early diagnostic biomarkers are urgently needed to distinguish CRC and AA from healthy control (HC). Alternative glycosylation of serum IgG has been shown to be closely associated with CRC. We aimed to explore the potential of IgG N-glycan as biomarkers in the early differential diagnosis of CRC. The study population was strictly matched to the exclusion criteria process. Serum IgG N-glycan profiles were analyzed by a robust and reliable relative quantitative method based on ultra-performance liquid chromatography (UPLC). Relative quantification and classification performance of IgG N-glycans were evaluated by Mann-Whitney U tests and ROC curve based on directly detected and derived glycan traits, respectively. Six and 14 directly detected glycan traits were significantly changed in AA and CRC, respectively, compared with HC. GP1 and GP3 were able to accurately distinguish AA from HC for early precancerous lesions screening. GP4 and GP14 provided a high value in discriminating CRC from HC. A novel combined index named GlycoF, including GP1, GP3, GP4, GP14 and CEA was developed to provide a potential early diagnostic biomarker in discriminating simultaneously AA (AUC = 0.847) and CRC (AUC = 0.844) from HC. GlycoF also demonstrated a superior CRC detection rate across CRC all stages and conspicuous prediction ability of risk of relapse. Serum IgG N-glycans analysis provided powerful early screening biomarkers that can efficiently differentiate CRC and AA from HC.

B-cell-specific ablation of ß-1,4-galactosyltransferase 1 prevents aging-related IgG glycans changes and improves aging phenotype in mice.

IgG N-glycans levels change with advancing age, making it a potential biomarker of aging. ß-1,4-galactosyltransferase (B4GALT) gene expression levels also increase with aging. Ultra performance liquid chromatography (UPLC) was used to examine changes inserum IgG N-glycans at six time points during the aging process. Most serum IgG N-glycans changed with aging in WT but not in CD19-cre B4GALT1 floxed mice. The relative abundance of fucosylated biantennary glycans with or without Neu5Gc structures changed with aging in heterozygous B4GALT1 floxed mice but not in homozygous B4GALT1 floxed mice. Additionally, the aging phenotype was more apparent in WT mice than in B4GALT1 floxed mice. These results demonstrate that fucosylated biantennary glycans and fucosylated biantennary glycans containing N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were affected by the B4GALT1 floxed mouse genotype. The changing levels of fucosylated monoantennary glycans observed with aging in WT mice was reversed in B4GALT1 floxed mice and was not sex specific. In summary, B-cell-specific ablation of B4GALT1 from a glycoproteomic perspective prevented age-related changes in IgG N-glycans in mice. SIGNIFICANCE: In this study, serum IgG glycoproteomic data in wild-type (WT) and B-cell-specific ablation of ß-1,4-galactosyltransferase 1 mice (B4GALT) were analyzed. Results showed that fucosylated biantennary glycans with or without N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were also affected by the B4GALT1 floxed mouse genotype. In terms of gender-specific information, the trend towards elevated fucosylated monoantennary glycans in WT mice was not seen in CD19-cre B4GALT1 floxed mice in either sex. B-cell-specific ablation of B4GALT1 plays an important role in age-related glycan changes; its specific functions and mechanisms are worthy of in-depth study. Our data suggest that investigating the relationship between galactosylation and aging may help advance the field of glycoproteomics and aging research.

The B-Cell-Specific Ablation of B4GALT1 Reduces Cancer Formation and Reverses the Changes in Serum IgG Glycans during the Induction of Mouse Hepatocellular Carcinoma.

Serum immunoglobulin G (IgG) glycosylation, especially galactosylation, has been found to be related to a variety of tumors, including hepatocellular carcinoma (HCC). However, whether IgG glycan changes occur in the early stages of HCC formation remains unclear. We found that the galactosylation level increased and that the related individual glycans showed regular changes over the course of HCC induction. Then, the effect of the B-cell-specific ablation of ß1,4galactosyltransferase 1 (CKO B4GALT1) and B4GALT1 defects on the IgG glycans that were modified during the model induction process and HCC formation is investigated in this study. CKO B4GALT1 reduces serum IgG galactosylation levels and reduces cancer formation. Furthermore, insignificant changes in the B-cell B4GALT1 and unchanged serum IgG galactosylation levels were found during cancer induction in female mice, which might contribute to the lower cancer incidence in female mice than in male mice. The gender differences observed during glycan and B4GALT1 modification also add more evidence that the B4GALT1 in B cells and in serum IgG galactosylation may play an important role in HCC. Therefore, the findings of the present research can be used to determine the methods for the early detection of HCC as well as for prevention.

Fucosylation of anti-dsDNA IgG1 correlates with disease activity of treatment-naïve systemic lupus erythematosus patients.

BACKGROUND: Systemic Lupus Erythematosus (SLE) is a complex and heterogeneous autoimmune disease mediated by quantities of autoantibodies in which anti-double-stranded DNA (anti-dsDNA) antibodies are important. Besides, glycosylation is one of the most commonly post-translational modifications of antibodies. The association of anti-dsDNA antibodies glycosylation and SLE disease activity is still unknown. METHODS: We enrolled 101 consecutive treatment-naïve SLE patients with positive anti-dsDNA antibodies from the Department of Rheumatology and Immunology at Ruijin Hospital, Shanghai, between 2017 and 2019. Serum samples were used in this study. We analysed the glycosylation of anti-dsDNA IgG and total IgG subclasses according to systemic lupus erythematosus disease activity index (SLEDAI) scores. Statistical analysis and machine learning were performed to assess the correlation between glycosylation of anti-dsDNA IgG and total IgG with disease activity. FINDINGS: Serum samples from 86 patients could be detected with anti-dsDNA IgG glycopeptide and subclass of IgG glycoform. Cluster analysis showed that glycosylation of anti-dsDNA IgG and total IgG subclasses were different in SLE patients. Fucosylation, galactosylation, and sialylation levels of anti-dsDNA IgG1 were increased with SLEDAI scores (all p<0.05). The results of machine learning showed that all the glycoforms of anti-dsDNA IgG1 had better performance with lower standardised square error (SSE) than that of total IgG1, with anti-dsDNA IgG1 fucosylation level having the lowest SSE (0.009). INTERPRETATION: Our study indicated that glycosylation of anti-dsDNA IgG was different from that of total IgG and fucosylation of anti-dsDNA IgG1 correlated best with SLE disease activity. FUNDING: This work is supported by the National Key Research and Development Program of China (2018YFC0910303), National Natural Science Foundation of China (81801592, 82101876), Clinical Research Plan of SHDC (SHDC2020CR4011), Ruijin Hospital Youth Incubation Project (KY2021607) and Shanghai Pujiang Young Rheumatologists Training Program (SPROG202006).

Characteristics of purified anti-ß2GPI IgG N-glycosylation associate with thrombotic, obstetric and catastrophic antiphospholipid syndrome.

OBJECTIVE: Anti-ß-2 glycoprotein I (anti-ß2GPI) antibodies, defined as primary pathogenic antibody in antiphospholipid syndrome (APS). It has been reported that IgG Fc N-glycosylation affects IgG effector, we aim to investigate the association of Fc glycosylation profiles of purified anti-ß2GP1 IgG with clinical features of APS. METHODS: We purify anti-ß2GPI IgG and total IgG from 82 APS patients including nine catastrophic antiphospholipid syndrome (CAPS) patients, as well as total IgG from 103 healthy controls to quantitatively analyse all detectable Fc N-glycanforms of all IgG subclasses with Multiple Reaction Monitoring (MRM) method based on UPLC-ESI-QqQ mass spectrometry. RESULTS: Both purified anti-ß2GPI IgG and APS total IgG showed altered N-glycan profiles when compared with healthy control (HC) IgG. Anti-ß2GPI IgG presented with lower galactosylation, increased bisection and core fucosylation compared with APS total IgG and HC IgG. We found higher galactosylation of aß2GPI IgG2 in thrombotic APS compared with the obstetric APS, and lower galactosylation of aß2GPI IgG2 associated with late pregnancy morbidity. Moreover, low galactosylation of all anti-ß2GPI IgG subclasses, increased bisection and core fucosylation of anti-ß2GPI IgG1/2 were strongly associated with CAPS and triple positivity of antiphospholipid antibodies (aPLs). CONCLUSION: We comprehensively characterize the N-Glycans landscape of both anti-ß2GP1 and total IgG in APS. Altered N-glycan profiles of anti-ß2GPI IgG enables enabled the antibodies with proinflammatory properties. Furthermore, we associated levels of IgG Fc-glycosylation with clinical features antiphospholipid syndrome. These findings could increase our understanding of anti-ß2GPI antibody mediated mechanisms in APS and be used to develop diagnostics and new target treatments.

Screening and diagnosis of colorectal cancer and advanced adenoma by Bionic Glycome method and machine learning.

Colorectal cancer (CRC), one of the major health problems worldwide, mostly develops from colorectal adenomas. Advanced adenomas are generally considered as precancerous lesions and patients are recommended to remove the adenomas. Screening for colorectal cancer is usually performed by fecal tests (FOBT or FIT) and colonoscopy, however, their benefits are limited by uptake and adherence. Most CRC develops from colorectal advanced adenomas, but there is currently a lack of effective noninvasive screening method for advanced adenomas. N-glycans in human serum hold the great potentials as biomarker for diagnosis of human cancers. Our aim was to discover blood-based markers for screening and diagnosis of advanced adenomas and CRC, and to ascertain their efficiency in classifying healthy controls, patients with advanced adenomas and CRC by incorporating machine learning techniques with reliable and simple quantitative method with "Bionic Glycome" as internal standard based on the high-throughput Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). The quantitative results showed that there is a positive correlation between multi-antennary, sialylated N-glycans and CRC progress, while bi-antennary core-fucosylated N-glycans are negatively correlated with CRC progress. Machine learning is a powerful classification tool, suitable for mining big data, especially the large amount of data generated by high-throughput technologies. Using the predictive model constructed by machine learning, we obtained the classification accuracy of 75% for classification of 189 samples including CRC, advanced adenomas and healthy controls, and the accuracy of 87% for detection of the disease group that required treatment, including CRC and advanced adenomas. To our delight, the model successfully applied to the prediction of 176 samples collected a few months later, and five samples were wrongly predicted in the disease group. Overall, this diagnostic model we constructed here has valuable potential in the clinical application of detecting advanced adenomas and colorectal cancer and could compensate for the limitations of the current screening methods for detection of CRC and advanced adenomas.

CA125-Tn ELISA assay improves specificity of pre-operative diagnosis of ovarian cancer among patients with elevated serum CA125 levels.

BACKGROUND: CA125 is the most widely used serum marker for preoperative diagnosis of ovarian cancer. However, CA125 elevation is not specific to ovarian cancer. More than 60% of patients who have elevated CA125 levels do not have ovarian cancer. To overcome the low specificity of CA125, we identified a CA125 glycoform that was specifically elevated in ovarian cancer and that may help in the further triage of patients with elevated serum CA125 levels. METHODS: We used antibody-lectin ELISA to detect various CA125 glycoforms. Among 21 lectins tested, VVA, a plant lectin that preferentially binds Tn antigen, showed significantly stronger binding with ovarian cancer-derived CA125 than benign condition-derived CA125. CA125-Tn levels were tested among patients with elevated CA125 levels (n=328, including 68 ovarian cancer, 15 ovarian borderline tumors, and 245 benign conditions). The efficacy of CA125-Tn in diagnosing ovarian cancer was evaluated using ROC analysis. RESULTS: Medians and 25th to 75th quartiles of CA125-Tn levels were 0.31 (0.18-0.65) in ovarian cancer, 0.07 (0.02-0.12) in ovarian borderline tumor, and 0.07 (0.01-0.12) in benign conditions. AUC of the ROC curve was 0.890 (95% CI: 0.845, 0.935) for CA125-Tn to discriminate ovarian cancer cases from nonmalignant cases (borderline tumors and benign conditions). Its performance was even better among patients older than 45 y (AUC: 0.905, 95% CI: 0.841, 0.969). Specificity was improved from 35.1% for CA125 to 75.7% for CA125-Tn among patients older than 45 y when sensitivity was fixed at 90%. CONCLUSIONS: CA125-Tn ELISA assay can improve specificity of the preoperative diagnosis of ovarian cancer and serve as a further triage strategy for patients with elevated CA125 levels.

Quantitation of sex-specific serum N-glycome changes in expression level during mouse aging based on Bionic Glycome method.

Studying the changes of serum N-glycome during mouse aging is beneficial to explore the molecular basis behind the alterations reported in human. However, such studies remainscarce and lack some information such as sialylation due to the method limitation. Here, we introduced Bionic Glycome method to quantify the serum N-glycome changes during C57BL/6 mouse aging (from the pubertal period to the old age stage). This technique enabled reliable and comprehensive quantitation of the expression level changes of more than 20 N-glycans in mouse serum at 12 time points in both genders for the first time, involving the analysis of sialic acid and its different linkages. The results demonstrated that the expression level of total glycans increased from middle age to old age. Interestingly, sex-specific N-glycome profiles and alterations were observed. Female mice showed higher level of serum fucosylation and lower level of serum afucosylation than male mice (fucosylation: p < 1.0E-6; afucosylation: p < 1.0E-6). Obviously, higher increase of serum fucosylation level was found in female mice than in male mice from middle age to old age. In addition, the opposite alterations of the afucosylated glycans with α2,3-linked sialic acid and those only with α2,6-linked sialic acid were observed at old age in male mice. These findings suggested that N-glycome could be a valuable target for investigating aging and possible contributors to aging.

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.

Profiling of IgG N-glycome during mouse aging: Fucosylated diantennary glycans containing one Neu5Gc-linked LacNAc are associated with age.

N-glycosylation of immunoglobulin G (IgG) has been reported to change in human aging and in some age-related diseases. To further understand the molecular processes that determine these alterations, a detailed examination of individual IgG N-glycans with aging remains required. Mouse is the most commonly used model animal in studies of aging and age-related diseases, and mice have the advantage of relatively controllable genetic and environment variations compared to human. In this study, we systemically investigated the changes in serum IgG N-glycome in C57BL/6 mice during aging at 12 time points (6-80 weeks) via ultraperformance liquid chromatography with fluorescence detection. The study demonstrated several important findings. First, four chromatographic IgG N-glycan peaks were identified for the first time, including a high-mannose glycan, a monoantennary glycan, and two afucosylated glycans. Second, most of the IgG glycan levels changed significantly and presented pronounced gender-related differences from 6 to 12 weeks. Interestingly, all the IgG glycan levels tended to be similar between male and female mice at 12 weeks. Third, the level of fucosylated diantennary glycans containing one N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) decreased gradually and showed a significant negative correlation with age from 24 to 80 weeks (r = -0.716, p < 0.0001), which was not sex-specific. SIGNIFICANCE: More comprehensive profile of murine IgG N-glycans by ultraperformance liquid chromatography with fluorescence detection was shown in this study with four newly identified chromatographic murine IgG N-glycan peaks. The majority of IgG N-glycans showed substantial stage-specific changes and sex-related differences during mouse aging, indicating a strict regulatory mechanism of glycan synthesis. The level of fucosylated diantennary glycans containing one Neu5Gc-linked LacNAc was significantly negatively correlated with age from 24 to 80 weeks, suggesting its great potential as an aging biomarker. The detailed characteristics of IgG N-glycosylation with aging in C57BL/6 mice demonstrated in the present study could provide essential reference data for studying the function and mechanism of IgG glycosylation in age-related researches based on C57BL/6 mouse models.

Correction to: The IgG galactosylation ratio is higher in spondyloarthritis patients and associated with the MRI score.

In the original version of the above article the References 19 and 20 were incorrect which cannot describe the development of the SPARCC score.

Dynamic alterations in serum IgG N-glycan profiles in the development of colitis-associated colon Cancer in mouse model.

BACKGROUND: Alternative glycosylation of serum IgG has been shown to be closely associated with colorectal cancer (CRC). Currently, a dynamic study which can not only minimize the influence of genetic background, environment and other interfering factors during cancer development, but also focus on investigating carcinogenic characteristics of IgG glycan is lacking. METHODS: Serum IgG N-glycans were characterized at four stages of CRC development by ultra-performance liquid chromatography in a typical colitis-related CRC mouse model induced by azoxymethane-dextran sodium sulfate. Furthermore, the expression of related glycosyltransferases in splenic B lymphocytes at the corresponding time was also assessed. RESULTS: The relative abundance of seven IgG glycans, which can be classified as monoantennary, core fucose, sialic acid, galactose and bisecting, was changed during tumor growth. The abundance of some glycans was altered during the first stage of cancer induction. Correspondingly, the expression of glycosyltransferases in splenic B lymphocytes and different tissues in cancer groups was also decreased compared to that in controls. CONCLUSIONS: This study represents the comprehensive analysis of IgG glycosylation in the dynamic process of colitis-associated CRC. To our knowledge, this is the first report that the expression of glycosyltransferases in mouse splenic B lymphocytes is consistent or inconsistent with the alterations of IgG N-glycans, and the variation tendency is tissue nonspecific. GENERAL SIGNIFICANCE: Providing a novel approach to identify the IgG glycans related to the development of CRC and laying a foundation for research on structure and function of glycans using mouse.

Dynamic analysis of N-glycomic and transcriptomic changes in the development of ovarian cancer cell line A2780 to its three cisplatin-resistant variants.

BACKGROUND: Platinum resistance development is a dynamic process that occurs during continuous chemotherapy and contributes to high mortality in ovarian cancer. Abnormal glycosylation has been reported in platinum resistance. Many studies on platinum resistance have been performed, but few of them have investigated platinum resistance-associated glycans based on N-glycomics. Moreover, glycomic alterations during platinum resistance development in ovarian cancer are rarely reported. Therefore, the objective of this study was to determine platinum resistance-related N-glycans in ovarian cancer cells during continuous exposure to cisplatin. These glycans might be involved in the mechanism of platinum resistance and serve as biomarkers to monitor its development. METHODS: This study mimicked the development of platinum resistance in ovarian cancer by continuously exposing A2780 cells to cisplatin. Cisplatin-resistant variants were confirmed by higher half maximal inhibitory concentration (IC50) values and increased P-glycoprotein (ABCB1, P-gp) expression compared to A2780 cells. Analysis of dynamic N-glycomic changes during the development of platinum resistance in cisplatin-resistant variants was performed with MALDI-time-of-flight (TOF)-MS combined with ethyl esterification derivatization, which were used to discriminate between α2,3- and α2,6-linkage N-acetylneuraminic acid. N-glycan alterations were further validated on a glycotransferase level via transcriptome sequencing and real-time PCR (RT-PCR). RESULTS: Compared to the A2780 cells, MS analysis indicated that α2,3-linked sialic structures and N-glycan gal-ratios were significantly higher, while fucosylated glycans were lower in three cisplatin-resistant variants. Transcriptome sequencing and RT-PCR showed that gene expression of ST3GAL6 and MGAT4A increased, while gene expression of FUT11, FUT1, GMDS, and B4GALT5 decreased in three cisplatin-resistant variants. CONCLUSIONS: Analysis of N-glycans and glycogene expression showed that α2,3-linked sialic structures might serve as biomarkers to monitor the development of platinum resistance and to guide individualized treatment of ovarian cancer patients.

Relative Quantitation of Subclass-Specific Murine IgG Fc N-Glycoforms by Multiple Reaction Monitoring.

N-Linked glycosylation of the fragment crystallizable (Fc) domain of immunoglobulin G (IgG) is considered a significant modulator of antibody functions, which is known to be subclass-specific. As mice are the most widely used model organisms in immunological research, determining the variation in Fc glycosylation among each murine IgG subclass in different physiological or pathological statuses is beneficial for studying how the IgG subclass effector function is affected by Fc glycosylation. In this study, we established a method to quantify murine IgG Fc glycoforms normalized to the protein abundance at a subclass-specific level for various mouse strains using multiple reaction monitoring. The glycoform level was normalized to the subclass protein abundance (subclass-specific peptide intensity) in each IgG subclass to eliminate the contribution from the subclass protein abundance. Both good linearity and high repeatability of the method were validated by investigating a mixed mouse serum sample. The method was applied to quantify the differences in subclass-specific IgG Fc N-glycoforms between systemic sclerosis (SSc) mice and healthy control mice. The results demonstrated that each IgG subclass had its own characteristic-altered glycosylation, implying the close association of subclass-specific IgG Fc glycosylation with SSc in mice. This report demonstrates a method with great reliability and practicality that has promising potential for the relative quantitation of subclass-specific IgG Fc N-glycoforms in multiple mouse models.

The IgG galactosylation ratio is higher in spondyloarthritis patients and associated with the MRI score.

OBJECTIVES: MRI is an important tool for evaluating inflammation levels and assessing treatment response in patients with ankylosing spondylitis (AS). However, it is expensive and requires experienced physicians. The goal of this study was to identify a biomarker correlated with the MRI score. METHODS: A total of 558 spondyloarthritis (SpA) patients including 527 AS patients, 10 psoriasis (PsA) patients, and 21 non-radiographic SpA (nr-SpA) patients and 725 controls were enrolled for the studies. Plasma IgG galactosylation (IgG-Gal) level was measured by mass spectrometry. Clinical indexes such as Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and C-reactive protein (CRP) were measured in all AS patients. MRIs and X-rays were obtained from 65 AS patients who were followed up for 6 months. RESULTS: The IgG-Gal ratio was twice as high in the AS patients compared with the controls. It correlated with inflammation indices which is evaluated by MRI according to SPARCC. (Pearson coefficient/p value was 0.6/7E10-6). In addition, AS patients with a higher IgG-Gal ratio at baseline tended to show greater improvement in inflammation scores by MRI both in 3-month follow-up and 6-month follow-up. CONCLUSION: The IgG-Gal ratio was significantly increased in AS patients. In clinical care, it may be used as a potential biomarker for diagnosis in the future. Key Points • IgG galactosylation level was abnormal in SpA patients. • IgG galactosylation level was associated with MRI indices.

Quantitative analysis of serum-based IgG agalactosylation for tuberculosis auxiliary diagnosis.

Tuberculosis (TB) is the leading infectious cause of mortality worldwide, especially in developing countries. However, effective means for TB diagnosis, especially for bacillus-negative (Bn) TB laboratory diagnosis, are urgently needed. In the present study, serum IgG from each tuberculosis patients and healthy controls was purified using affinity chromatography. The samples were then analyzed using mass spectrometry (MS) and ultraperformance liquid chromatography (UPLC) methods. We quantitatively assessed the changes of serum IgG galactosylation in 567 human serum samples including 377 pulmonary TB patients and 190 healthy donors (HDs). We found significantly more agalactosylated (G0) vs monogalactosylated (G1) and digalactosylated (G2) N-glycans of IgG in TB patients, including smear-negative TB patients, than in HDs. The detection rate of TB diagnostic performance by MS for IgG-Gal ratio G0/(G1 + G2 × 2) is 90.48% for bacillus-positive (Bp) and 73.16% for Bn TB patients. Further, combination of MS method with other routine laboratory TB diagnostic methods significantly increased the detection rate to 91.01%-98.39%. Similar results were observed in Mycobacterium tuberculosis (M. tb) infection mouse models. The decrease in galactosylation of IgG in TB patients was also qualitatively confirmed using specific lectin blot assay. Using the above techniques, we can discriminate the content of IgG G0 with terminal N-acetylglucosamine and IgG-Gal ratio G0/(G1 + G2 × 2) between TB patients and HDs. Our data suggest that quantitative analysis of serum-based IgG-Gal ratio G0/(G1 + G2 × 2) could be used for TB auxiliary diagnosis with high effectiveness and feasibility and its combination with other routine laboratory TB diagnostic methods could remarkably improve the detection rate.

Use of the serum glycan state to predict ovarian cancer patients' clinical response to chemotherapy treatment.

Ovarian cancer is the most lethal gynecologic carcinoma; because the tumor often relapses shortly after treatment. Glycosylation plays important roles in cancer drug resistance and could be used as biomarkers to predict the drug response of patients. We used MALDI-QIT-TOF MS to analyze the serum glycomic from patients with different drug responses. Samples were collected before treatment; follow-up visit were performed after 6 months. Forty-eight drug-sensitive patients and 16 drug-resistant patients were enrolled. Compared with drug-sensitive patients, 5 glyco-subclasses and 5 single glycans were significantly altered in drug-resistant patients. Lewis type, α2,3 sialic acid and multibranch glycans were increased, α2,6 sialic acid glycans were decreased. The peak at m/z 2986.44 showed stronger prediction abilities than other single glycans, with an AUC of 0.83. A panel of three increased glycans (m/z 2401.36, H5N4F1S2, a Lewis type biantennary glycan; m/z 2986.44, H6N5S3, a triantennary trisialylated glycan; m/z 3086.39, H6N5F1S3, a Lewis type triantennary glycan) combined with CA125 achieved an AUC value of 0.88, showing a strong discrimination performance. This study provides new insights into N-glycosylation patterns in ovarian cancer patients with different drug response. These altered glycans might serve as biomarkers to reflect patients' drug sensitivity and to guide clinical treatment. SIGNIFICANCE: A large number of ovarian cancer patients experience tumor relapse shortly after initial treatment. Glycosylation plays important roles in cancer drug resistance and could be used as a biomarker to predict the drug response of patients. However, the glycosylation expressed in patients with different drug response have not been elucidated. In the present study, we used MALDI-QIT-TOF MS to analyze the serum glycomic levels of patients with different drug responses. Several glycans were changed significantly between these two groups. A panel of three increased glycans (m/z 2401.36, a Lewis type biantennary glycan, 2986.44, a triantennary trisialylated glycan, and 3086.39, a Lewis type triantennary glycan) combined with CA125 performed better descrimination of these two groups with AUC of 0.88. These altered glycans might serve as biomarkers to reflect patients' drug sensitivity and to guide clinical treatment.

Prediction of neoadjuvant chemotherapeutic efficacy in patients with locally advanced gastric cancer by serum IgG glycomics profiling.

BACKGROUND: Neoadjuvant chemotherapy (NACT) could improve prognosis and survival quality of patients with local advanced gastric cancer (LAGC) by providing an opportunity of radical operation for them. However, no effective method could predict the efficacy of NACT before surgery to avoid the potential toxicity, time-consuming and economic burden of ineffective chemotherapy. Some research has been investigated about the correlation between serum IgG glycosylation and gastric cancer, but the question of whether IgG glycome can reflect the tumor response to NACT is still unanswered. METHOD: Serum IgG glycome profiles were analyzed by Ultra Performance Liquid Chromatography in a cohort comprised of 49 LAGC patients of which 25 were categorized as belonging to the NACT response group and 24 patients were assigned to the non-response group. A logistic regression model was constructed to predict the response rate incorporating clinical features and differential N-glycans, while the precision of model was assessed by receiver operating characteristic (ROC) analysis. RESULTS: IgG N-glycome analysis in pretreatment serum of LAGC patients comprises 24 directly detected glycans and 17 summarized traits. Compared with IgG glycans of non-response group, agalactosylated N-glycans increased while monosialylated N-glycans and digalactosylated N-glycans decreased in the response group. We constructed a model combining patients' age, histology, chemotherapy regimen, GP4(H3N4F1), GP6(H3N5F1), and GP18(H5N4F1S1), and ROC analysis showed this model has an accurate prediction of NACT response (AUC = 0.840) with the sensitivity of 64.00% and the specificity of 100%. CONCLUSION: We here firstly present the profiling of IgG N-glycans in pretreatment serum of LAGC. The alterations in IgG N-glycome may be personalized biomarkers to predict the response to NACT in LAGC and help to illustrate the relationship between immunity and effect of NACT.