A highly specific antibody against the core fucose of the N-glycan in IgG identifies the pulmonary diseases and its regulation by CCL2.

Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

Salivary L-fucose as a biomarker for oral potentially malignant disorders and oral cancer.

BACKGROUND: The objective of this study was to evaluate the serum and salivary L-fucose in oral potentially malignant disorders (OPMDs) and oral cancer (OC) in order to investigate the possibility of using this as biomarker for early diagnosis. MATERIALS AND METHODS: The study included 85 participants, who were grouped as control (30), OPMDs patients (25), and OC patients (30). Serum and unstimulated whole saliva were collected from participants of all groups and fucose estimation was done using spectrophotometry. The results were tabulated and analyzed statistically. RESULTS: The mean serum L-fucose levels in normal, OPMDs, and OC group were 3.49, 19.18, and 35.75 mg/dl, respectively, while the levels of salivary L-fucose were 3.18, 7.02, and 11.66 mg/dl, respectively. A highly significant rise (P < 0.001) in serum and salivary L-fucose was observed in the study participants compared to control. CONCLUSIONS: The present study showed a significant and gradual increase in serum and salivary L-fucose from control to OPMDs to OC. From this study, we suggest that L-fucose can be used as a reliable biomarker and saliva can be used as a diagnostic fluid for screening and early detection of OC.

Identification of fucosylated Fetuin-A as a potential biomarker for cholangiocarcinoma.

PURPOSE: Cholangiocarcinoma (CCA) is a malignancy of the bile ducts. The purpose of this discovery study was to identify effective serum markers for surveillance of cholangiocarcinoma. EXPERIMENTAL DESIGN: Using a glycomic method, patients with CCA were determined to have increased levels of alpha-1,3 and alpha-1,6 linked fucosylated glycan. Proteomic analysis of the serum fucosylated proteome identified proteins such as alpha-2-macroglobulin, kininogen, hemopexin, fetuin-A, alpha-1 anti-trypsin, and ceruloplasmin as being hyperfucosylated in HCC. The levels of these glycoproteins in 109 patients with CCA, primary sclerosing cholangitis (PSC), and control patients were compared to the performance of CA-19-9, the current "gold standard" assay for cholangiocarcinoma. RESULTS: Fucosylated Fetuin-A (fc-Fetuin-A) had the best ability to differentiate CCA from PSC, with an AUROC of 0.812 or 0.8665 at differentiating CCA from those with PSC or other liver disease. CA-19-9 had poor ability to differentiate PSC from cholangiocarcinoma (AUROC of 0.625). CONCLUSION AND CLINICAL RELEVANCE: Using glycomic and proteomic methods we identified a set of proteins that contain altered glycan in the sera of those with CCA. One of these proteins, fucosylated Fetuin-A may have value in the surveillance of people at risk for the development of cholangiocarcinoma.

Increased α1-3 fucosylation of α-1-acid glycoprotein (AGP) in pancreatic cancer.

Pancreatic cancer (PDAC) lacks reliable diagnostic biomarkers and the search for new biomarkers represents an important challenge. Previous results looking at a small cohort of patients showed an increase in α-1-acid glycoprotein (AGP) fucosylation in advanced PDAC using N-glycan sequencing. Here, we have analysed AGP glycoforms in a larger cohort using several analytical techniques including mass spectrometry (MS), capillary zone electrophoresis (CZE) and enzyme-linked lectin assays (ELLAs) for determining AGP glycoforms which could be PDAC associated. AGP from 31 serum samples, including healthy controls (HC), chronic pancreatitis (ChrP) and PDAC patients, was purified by immunoaffinity chromatography. Stable isotope labelling of AGP released N-glycans and their analysis by zwitterionic hydrophilic interaction capillary liquid chromatography electrospray MS (µZIC-HILIC-ESI-MS) showed an increase in AGP fucosylated glycoforms in PDAC compared to ChrP and HC. By CZE-UV analysis, relative concentrations of some of the AGP isoforms were found significantly different compared to those in PDAC and HC. Finally, ELLAs using Aleuria aurantia lectin displayed a significant increase in AGP fucosylation, before and after AGP neuraminidase treatment, in advanced PDAC compared to ChrP and HC, respectively. Altogether, these results indicate that α1-3 fucosylated glycoforms of AGP are increased in PDAC and could be potentially regarded as a PDAC biomarker.

Comparison of Serum Fucose Levels in Leukoplakia and Oral Cancer Patients.

BACKGROUND: Tumor markers, designated as a broad group of substances produced by malignancies, could be in the form of biochemical substances, immunological substances, cell surface changes and genetic alterations. Cancer, a disorder of cellular behavior is characterized by alteration of serum glycoproteins. L-fucose, a hexose, which is the terminal sugar in most of the plasma glycoproteins, may be useful as a tumor marker for the detection, monitoring and prognostic assessment of malignancies. The aim of the study was to ascertain the role of serum fucose as a biomarker for early detection of oral cancer and to compare serum fucose levels in healthy controls, leukoplakia and oral cancer patients. MATERIALS AND METHODS: The study included 60 (100.0%) subjects, who were grouped as 20 (33.3%) control subjects, 20 (33.3%) squamous cell carcinoma patients and 20 (33.3%) leukoplakia patients. Fucose estimation was done using UV-visible spectrophotometry based on the method as adopted by Winzler using cysteine reagent. The results were analyzed statistically using ANOVA with Bonferroni post hoc tests. RESULTS: Results showed a high significance in serum fucose in oral squamous cell carcinoma (OSCC) and leukoplakia subjects compared to normal controls. There was a gradual increase in the values noted from control to leukoplakia and to squamous cell carcinoma. CONCLUSIONS: Estimation of serum fucose may be a reliable marker and can be used as an effective diagnostic biomarker in oral squamous cell carcinoma patients.

ESI-LC-MS Method for Haptoglobin Fucosylation Analysis in Hepatocellular Carcinoma and Liver Cirrhosis.

A method for the detection of fucosylated glycans from haptoglobin in patient serum has been developed that provides enhanced sensitivity. The workflow involves isolation of the haptoglobin using an HPLC-based affinity column followed by glycan removal, extraction, and desialylation. The fucosylated glycans are then derivatized by Meladrazine, which significantly enhances the detection of the glycans in electrospray ionization. The separation of the derivatized glycans in a HILIC column shows that eight glycans from haptoglobin can be detected using less than 1 µL of a serum sample, with excellent reproducibility and quantitation, where without derivatization the glycans could not be detected. The ratio of the fucosylated peaks to their corresponding nonfucosylated forms shows that the fucosylated glycans are upregulated in the case of hepatocellular carcinoma (HCC) samples versus cirrhosis samples, where the relatively low abundance bifucosylated tetra-antennary form can be detected and may be a particularly good marker for HCC.

Relative Quantification and Higher-Order Modeling of the Plasma Glycan Cancer Burden Ratio in Ovarian Cancer Case-Control Samples.

An early-stage, population-wide biomarker for ovarian cancer (OVC) is essential to reverse its high mortality rate. Aberrant glycosylation by OVC has been reported, but studies have yet to identify an N-glycan with sufficiently high specificity. We curated a human biorepository of 82 case-control plasma samples, with 27%, 12%, 46%, and 15% falling across stages I-IV, respectively. For relative quantitation, glycans were analyzed by the individuality normalization when labeling with glycan hydrazide tags (INLIGHT) strategy for enhanced electrospray ionization, MS/MS analysis. Sixty-three glycan cancer burden ratios (GBRs), defined as the log10 ratio of the case-control extracted ion chromatogram abundances, were calculated above the limit of detection. The final GBR models, built using stepwise forward regression, included three significant terms: OVC stage, normalized mean GBR, and tag chemical purity; glycan class, fucosylation, or sialylation were not significant variables. After Bonferroni correction, seven N-glycans were identified as significant (p < 0.05), and after false discovery rate correction, an additional four glycans were determined to be significant (p < 0.05), with one borderline (p = 0.05). For all N-glycans, the vectors of the effects from stages II-IV were sequentially reversed, suggesting potential biological changes in OVC morphology or in host response.

Plasma N-Glycome Signature of Down Syndrome.

In recent years, plasma N-glycans have emerged as biomarkers for health and disease. Here, we studied N-glycomic changes in Down Syndrome (DS). Because of the progeroid phenotype of DS, we focused on the dissection of syndrome- and aging-associated glycomic changes, as well as the interaction thereof. We analyzed the plasma N-glycome of 76 DS persons, 37 siblings (DSS), and 42 mothers (DSM) of DS persons by DNA-sequencer-aided, fluorophore-assisted-carbohydrate-electrophoresis, as well as by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). The results showed an overall decrease of galactosylation and α2,3 sialylation, a concomitant increase of the level of fucosylated N-glycans as well as of monogalactosylated diantennary N-glycans in DS, while the GlycoAgeTest and the ratio of the two core-fucosylated, monogalactosylated diantennary isomers (galactose positioned on α1,6 arm versus α1,3 arm) were the strongest DS discriminators. Hypogalactosylation is a characteristic of both DS and aging of control individuals. A decrease in α2,3-sialylated species is also common to DS and aging of controls. However, regarding to α2,6-sialylated tri- and tetragalactosylated N-glycan species, we found those to be lowered in DS but showed an increase with age in the same persons, while these glycans were not affected by aging in control individuals. In conclusion, we identified specific glycomic changes associated with DS, aging in DS, as well as aging in controls, identifying glycomic features in line with accelerated aging in DS. Notably, our data demonstrate an aging phenotype in DS which only in part overlaps with aging in controls but reveals DS-specificity.

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D.

BACKGROUND: RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with FcγRIII, with less Fc-fucosylation strengthening the interaction. STUDY DESIGN AND METHODS: Anti-D IgG1 Fc-glycosylation patterns in 93 plasma samples from 28 male and 28 female Dutch HIDs and RhIG were analyzed with mass spectrometry. The Fc-glycosylation profiles of HIDs were evaluated with regard to their immunization history. RESULTS: HID sera demonstrated clearly lowered anti-D Fc-fucosylation compared to normal IgG fucosylation (93%); this was more pronounced for female than for male HIDs (47% vs. 65%, p = 0.001). RhIG preparations from seven manufacturers varied greatly in the level of Fc-fucosylation (56%-91%). The level of fucosylation slightly increased upon repeated immunization, although it remained fairly constant over time. The RhIG from the different manufacturers all demonstrated increased Fc-galactosylation (64%-82%) compared to total IgG (38%-51%). CONCLUSION: RhIG preparations vary in Fc-fucosylation and all demonstrate increased galactosylation. Despite not knowing the exact working mechanism, immunoprophylaxis could perhaps be optimized by selection of donors whose anti-D have low amounts of Fc-fucose, to increase the clearance activity of anti-D preparations, as well as high amounts of galactosylation, for anti-inflammatory effects. Implementing a biologic assay in the standardization of RhIG preparations might be considered.

Dietary fucoidan of Acaudina molpadioides and its enzymatically degraded fragments could prevent intestinal mucositis induced by chemotherapy in mice.

Mucositis is a common problem that results from cancer chemotherapy and is a cause of significant morbidity and occasional mortality. Its prevention and successful treatment can significantly enhance the quality of life of patients and improve their survival. Sea cucumber is a traditional aquatic food that has both nutritional and medicinal value. The polysaccharide fucoidan from the sea cucumber (SC-FUC) has various bioactivities. We examined the protective effect of different molecular weights (MWs 50 kDa-500 kDa) of fucoidan from the sea cucumber, Acaudina molpadioides, in a mouse model of cyclophosphamide (Cy)-induced intestinal mucositis. Results showed that the oral administration of SC-FUC markedly reversed Cy-induced damage in the mice. The sea cucumber fucoidan notably increased the ratio of the length of the intestinal villus to the crypt depth and ameliorated the IFN-γ/IL-4 ratio that signifies Th1/Th2 immune balance. Moreover, all the fucoidans in this study enhanced the expression of IgA by accelerating the expression of IL-6 that is probably combined with IL-10. The differing effects of the varied molecular weights of fucoidan may be due to the difference in the efficiency of absorption. This is a novel study on the potential preventive effects of SC-FUC on intestinal mucositis that may be related to the efficiency of its absorption during digestion. Sea cucumber fucoidan (SC-FUC) may be used as a potential food supplement to prevent chemotherapeutic mucositis.

Inflammation, oxidative stress and L-fucose as indispensable participants in schistosomiasis-associated colonic dysplasia.

BACKGROUND: Schistosomiasis is a parasitic disease causing chronic ill health in humans with a serious consequences for socio-economic development in tropical and subtropical regions. There is also evidence linking Schistosoma mansoni to colonic carcinoma occurrence. The aim of this study was to evaluate some inflammatory and oxidative stress biomarkers, as well as L-fucose as linkers between intestinal schistosomiasis and colonic dysplasia development in mice. MATERIALS AND METHODS: This study was conducted upon 80 mice that were divided the control group (10 non infected mice) and infected group which was subdivided into 7 sub-groups (10 mice each) according to the time of sacrifaction in the post infection (p.i.) period, 10 mice being sacrificed every two weeks from 6 weeks p.i. to 18 weeks p.i. Tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), and pentraxin 3 (PTX3) levels were estimated by immunoassay. The L-fucose level, and thioredoxin reductase (TrxR) and lactate dehydrogenase (LDH) activities were also evaluated in colonic tissue. RESULTS: The current study revealed statistically significant elevation in the studied biochemical markers especially at 16 and 18 weeks p.i. The results were confirmed by histopathological examination that revealed atypical architectural and cytological changes in the form of epithelial surface serration and nuclear hyper-chromatizia at 14, 16 and 18 weeks p.i. CONCLUSIONS: inflammation, oxidative stress and L-fucose together may form an important link between Schistosomal mansoni infection and colonic dysplasia and they can be new tools for prediction of colonic dysplasia development in experimental schistosomiasis.

Targeting deeper the human serum fucome by a liquid-phase multicolumn platform in combination with combinatorial peptide ligand libraries.

Combinatorial peptide ligand library (CPLL) was evaluated as an off line step to narrow the differences of protein concentration in human serum prior to the capturing of human fucome from disease-free and breast cancer sera by a multicolumn platform via lectin affinity chromatography (LAC) followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two monolithic lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) columns were utilized to capture the fucome, which was subsequently fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to tryptic digestion prior to LC-MS/MS analysis. AAL has a strong affinity towards core fucosylated N-glycans and has a weak binding towards fucose in the outer arm while LTA can bind to glycans having fucose present in the outer arm. The combined strategy consisting of the CPLL, multicolumn platform and LC-MS/MS analysis permitted the identification of the differentially expressed proteins (DEPs) in breast cancer serum yielding 58 DEPs in both the LTA and AAL fractions with 6 DEPs common to both lectins. 17 DEPs were of the low abundance type, 16 DEPs of the borderline abundance type, 4 DEPs of the medium abundance type and 15 DEPs of the high abundance type. The remaining 6 DEPs are of unknown concentration. Only proteins exhibiting 99.9% protein identification probability, 95% peptide identification probability, and a minimum of 5 unique peptides were considered in finding the DEPs via scatterplots.

Dynamic changes in glycosylation and glycan composition of serum FSH and LH during natural ovarian stimulation.

BACKGROUND: Glycosylation and glycan composition are of fundamental importance for the biological properties of FSH and LH. The aim of this study was to determine the glycosylation, sialylation, and sulfonation of serum FSH and LH throughout the normal menstrual cycle. METHODS: Serum samples were collected from 79 healthy women with regular menstrual cycles. The mean numbers of anionic monosaccharide (AMS), sialic acid (SA), and sulfonated N-acetylgalactosamine (SU) residues per FSH and LH molecule were estimated for all sera with methods based on electrophoreses, neuraminidase treatments, and fluoroimmunoassays of the gonadotrophins. RESULTS: Di-glycosylated glycoforms (FSHdi, LHdi) were detected in serum in addition to tetra-glycosylated FSH (FSHtetra) and tri-glycosylated LH (LHtri). FSHdi exhibited two peaks: one on day 5 to 7 and one, more pronounced, at midcycle. FSHtetra plateaued at a high concentration from day 5 to 15, without a midcycle peak. There were lower concentrations of LHdi than LHtri, except at midcycle when the opposite occurred. The mean numbers of SA and SU residues per molecule of FSH and LH in serum showed four different patterns during the cycle, all with highly significant (P < 0.0001) differences between levels at different phases of the cycle. The pattern of SA residues on FSH was 'M'-shaped, and that of SU on LH 'V'-shaped. CONCLUSION: Serum FSH and LH governing the natural ovarian stimulation process exhibited dynamic changes of glycosylation and glycan composition. This new information on the FSH and LH molecular structures may lead to more successful mono-ovulatory treatment regimens for ovulation induction in anovulatory women.

Targeting human serum fucome by an integrated liquid-phase multicolumn platform operating in "cascade" to facilitate comparative mass spectrometric analysis of disease-free and breast cancer sera.

A fully integrated platform was developed for capturing/fractionating human fucome from disease-free and breast cancer sera. It comprised a multicolumn operated by HPLC pumps and switching valves for the simultaneous depletion of high abundance proteins via affinity-based subtraction and the capturing of fucosylated glycoproteins via lectin affinity chromatography followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) were utilized. The platform allowed the "cascading" of the serum sample from column-to-column in the liquid phase with no sample manipulation between the various steps. This guaranteed no sample loss and no propagation of experimental biases between the various columns. Finally, the fucome was fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to trypsinolysis for LC-MS/MS analysis. This permitted the identification of the differentially expressed proteins (DEP) in breast cancer serum yielding a broad panel of 35 DEP from the combined LTA and AAL captured proteins and a narrower panel of eight DEP that were commonly differentially expressed in both LTA and AAL fractions, which are considered as more representative of cancer altered fucome.

Protective effect of Cardiospermum halicacabum leaf extract on glycoprotein components on STZ-induced hyperglycemic rats.

OBJECTIVE: To investigate the protective role of Cardiospermum halicacabum (C. halicacabum) leaf extract on glycoprotein metabolism in streptozotocin (STZ)-induced diabetic rats. METHODS: Diabetes was induced in male albino Wistar rats by intraperitonial administration of STZ. The C. halicacabum leaf extract (CHE) was administered orally to normal and STZ-diabetic rats for 45 days. The effects of C. halicacabum leaf extract (CHE) on plasma and tissue glycoproteins (hexose, hexosamine, fucose and sialic acid) were determined. RESULTS: The levels of plasma and tissues glycoproteins containing hexose, hexosamine and fucose were significantly increased in STZ-induced diabetic rats. In addition, the level of sialic acid significantly increased in plasma and liver while decreased in kidney of STZ-induced diabetic rats. After administration of CHE to diabetic rats, the metabolic alteration of glycoprotein reverted towards normal levels. CONCLUSIONS: The present study indicates that the CHE possesses a protective effect on abnormal glycoprotein metabolism in addition to its antihyperglycemic activity.

Identification and confirmation of differentially expressed fucosylated glycoproteins in the serum of ovarian cancer patients using a lectin array and LC-MS/MS.

In order to discover potential glycoprotein biomarkers in ovarian cancer, we applied a lectin array and Exactag labeling based quantitative glycoproteomics approach. A lectin array strategy was used to detect overall lectin-specific glycosylation changes in serum proteins from patients with ovarian cancer and those with benign conditions. Lectins, which showed significant differential response for fucosylation, were used to extract glycoproteins that had been labeled using isobaric chemical tags. The glycoproteins were then identified and quantified by LC-MS/MS, and five glycoproteins were found to be differentially expressed in the serum of ovarian cancer patients compared to benign diseases. The differentially expressed glycoproteins were further confirmed by lectin-ELISA and ELISA assay. Corticosteroid-binding globulin (CBG), serum amyloid p component (SAP), complement factor B (CFAB), and histidine-rich glycoprotein (HRG) were identified as potential markers for differentiating ovarian cancer from benign diseases or healthy controls. A combination of CBG and HRG (AUC = 0.825) showed comparable performance to CA125 (AUC = 0.829) in differentiating early stage ovarian cancer from healthy controls. The combination of CBG, SAP, and CA125 showed improved performance for distinguishing stage III ovarian cancer from benign diseases compared to CA125 alone. The ability of CBG, SAP, HRG, and CFAB to differentiate the serum of ovarian cancer patients from that of controls was tested using an independent set of samples. Our findings suggest that glycoprotein modifications may be a means to identify novel diagnostic markers for detection of ovarian cancer.

Fragmentation and site-specific quantification of core fucosylated glycoprotein by multiple reaction monitoring-mass spectrometry.

Glycosylation modifications of proteins have been attracting increasing attention due to their roles in the physiological and pathological processes of the cell. Core fucosylation (CF), one special type of glycan structure in glycoproteins, has been linked with tumorigenesis. The study of protein glycosylation has been hindered by the technical challenges caused by the microheterogeneity of glycan modifications. In commonly used methods, sugar chains on the peptide were released using endoglycosidase, and the glycan and peptides were analyzed separately with mass spectrometry. Although mass spectrometric analysis can be performed easily in this way, an increase in false positives when assigning glycosites was inevitable. Our earlier research demonstrated a strategy combining Endo F3-catalyzed partial deglycosylation with MS(3) (MS/MS/MS) scanning triggered by the neutral loss of a fucose to precisely identify CF proteins on a large scale. In this research, fragmentations of partially deglycosylated glycopeptides were studied using a triple quadrupole mass spectrometer, and a quantification method that coupled our published identification strategy with multiple reaction monitoring-mass spectrometry (MRM-MS) analysis was developed to obtain site-specific quantification information of core fucosylated peptides. To illustrate the feasibility of the quantification method, the CF peptides of target proteins in clinical serum were quantified and compared as a preliminary demonstration.

Quantitation of plasma 13C-galactose and 13C-glucose during exercise by liquid chromatography/isotope ratio mass spectrometry.

The utilisation of carbohydrate sources under exercise conditions is of considerable importance in performance sports. Incorporation of optimal profiles of macronutrients can improve endurance performance in athletes. However, gaining an understanding of the metabolic partitioning under sustained exercise can be problematical and isotope labelling approaches can help quantify substrate utilisation. The utilisation of oral galactose was investigated using (13)C-galactose and measurement of plasma galactose and glucose enrichment by liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). As little as 100 µL plasma could readily be analysed with only minimal sample processing. Fucose was used as a chemical and isotopic internal standard for the quantitation of plasma galactose and glucose concentrations, and isotopic enrichment. The close elution of galactose and glucose required a correction routine to be implemented to allow the measurement, and correction, of plasma glucose δ(13)C, even in the presence of very highly enriched galactose. A Bland-Altman plot of glucose concentration measured by LC/IRMS against glucose measured by an enzymatic method showed good agreement between the methods. Data from seven trained cyclists, undergoing galactose supplementation before exercise, demonstrate that galactose is converted into glucose and is available for subsequent energy metabolism.

Human serum N-glycan profiles are age and sex dependent.

BACKGROUND: protein glycosylation varies with the physiological and pathological status of the cell. Consequently, analysis of protein-linked glycans has growing importance both in basic glycobiological research and as a potential tool for monitoring the physiological state in humans. DESIGN, SETTING AND PARTICIPANTS: a total of 265 healthy northern Chinese men and women were grouped by age and gender. The mean age in males and females was similar. OBJECTIVE: the study is aimed to evaluate the effects of the age and gender on the human serum N-glycans profiles in the clinical diagnose of ageing and disease. METHODS: the 265 human serum N-glycan profiles were obtained by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Comparison of N-glycan profiles was carried out among the different genders and age groups and the data were analysed with the GeneMapper software. RESULTS: age-related changes in the three N-glycan structures (NGA2F, NGA2FB and NA2F) were observed. Interestingly, fucosylation of N-glycans was significantly different (P < 0.0001) between men and women: more core-α-1,6-fucosylated glycans were detected in women, whereas more branching-α-1,3-fucosylated N-glycans were seen in men. CONCLUSIONS: the N-glycome profile in serum is gender and age dependent. This should be taken into consideration in the development of serum glycome markers.

Fucosylated glycoproteomic approach to identify a complement component 9 associated with squamous cell lung cancer (SQLC).

Human lung cancer is a major cause of cancer mortality worldwide. Understanding the pathophysiological features and the development of novel biomarkers for diagnosis as well as treatment are major tasks. In the present study, sera from ten SQLC patients and healthy control (HEC) were collected and pooled, respectively. The pooled sera were depleted via an immunoaffinity method and further subjected to fucosylation enrichment. Enriched fucosylated glycoproteins were resolved by SDS-PAGE and subsequently analyzed by LC-ESI-MS/MS. From comparative proteomic analysis, we selected the C9 protein. C9 protein levels were validated by Western blot, protein arrays and the fucosylation levels of C9 by hybrid lectin ELISA (HLE) in the sera of 120 HEC and 118 SQLC patients. The C9 protein level was 6.4-fold higher in SQLC patients compared to HEC, as determined by Western blot analysis. The results were concurrently confirmed by a protein array that showed a C9 level significantly higher in SQLC patients, as compared to HEC, with a sensitivity of 53% and a specificity of 89%. C9 fucosylation levels were significantly higher in SQLC patients compared to HEC (p<0.05) when tested by HLE. These findings suggest that C9 and fucosylated form could serve as a useful marker for SQLC.