Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age-and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels.

Glycosylation is a diverse but critically important post-translational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest. In the current study, the significant improvements in efficiency, selectivity, and analysis speed offered by ultra performance liquid chromatography (UPLC) profiling of fluorescently labeled N-linked oligosaccharides on a recently introduced sub-2 μm hydrophilic interaction (HILIC) based stationary phase are demonstrated to identify cancer associated alterations in the serum N-glycome of patients bearing stomach adenocarcinoma. The contribution of the glycosylation present on four highly abundant serum proteins namely, IgG, haptoglobin, transferrin, and α1-acid glycoprotein was evaluated. Alterations in the glycosylation present on these four proteins isolated from the pathologically staged cancer serum using either affinity purification or two-dimensional electrophoresis were then investigated as possible markers for stomach cancer progression. In agreement with previous reports, an increase in sialylation was observed on haptoglobin, transferrin, and α1-acid glycoprotein in the cancerous state. Increased levels of core fucosylated biantennary glycans and decreased levels of monogalactosylated core fucosylated biantennary glycans were present on IgG with increasing disease progression. The speed and selectivity offered by the sub-2 μm HILIC phase make it ideal for rapid yet highly efficient separation of complex oligosaccharide mixtures such as that present in the serum N-glycome.

Prostate cancer is the most common solid organ malignancy affecting men in the United States and Western Europe. Currently, the main diagnostic tools used to look for evidence of prostate cancer include physical examination using digital rectal exam (DRE), serum concentrations of prostate specific antigen (PSA) and biopsy. However, due to the low specificity of PSA in differentiating prostate cancer from other benign conditions, many patients undergo overtreatment for their disease. There is an urgent need for additional markers to improve the diagnostic accuracy for early stages of prostate cancer. Proteomic analysis of serum has the potential to identify such markers. An initial discovery study has been completed using 12 serum samples from patients with different grades of prostate cancer (Gleason score 5 and 7) undergoing radical prostatectomy. Serum samples were subjected to immunoaffinity depletion and protein expression analysis using 2D-DIGE. Image analysis isolated 63 spots that displayed differential expression between the Gleason score 5 and 7 cohorts (p < 0.05), 13 of which were identified as statistically significant using two independent image analysis packages. Identification of differentially expressed spots was carried out using LC-MS/MS. Because of their functional relevance and potential significance with regards to prostate cancer progression, two of these proteins, pigment epithelium-derived factor (PEDF) and zinc-alpha2-glycoprotein (ZAG), have undergone extensive validation in serum and tissue samples from the original cohort and also from a larger independent cohort of patients. These results have indicated that PEDF is a more accurate predictor of early stage prostate cancer. We are confident that proteomics-based approaches have the potential to provide more insight into the underlying molecular mechanisms of the disease and also hold great promise for biomarker discovery in prostate cancer.

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.

Despite the reduced incidence of gastric cancer in the developed world, a diagnosis of stomach carcinoma still carries a poor prognosis due to the asymptomatic nature of the disease in the early stages, subsequent advanced stage diagnosis, and a low 5 year survival rate. Endoscopy remains the primary standard for diagnosis of stomach carcinoma and the current marker, carbohydrate antigen 19-9 (CA19-9) lacks the levels of sensitivity and specificity required in order to make it clinically useful for diagnostic monitoring. Therefore, there is a current need for additional markers to improve the diagnostic accuracy for the early stages of stomach cancer. Together, glycomic, proteomic, and glycoproteomic analyses of serum have the potential to identify such probable markers. A discovery study is reported here using preoperative serum from 80 stomach cancer patients, 10 patients bearing benign stomach disease, and 20 matched controls. Glycomic analysis of the total and immunoaffinity depleted serum revealed statistically significant increases in the levels of sialyl Lewis X epitopes (SLe(X)) present on triantennary glycans accompanied by increased levels of core fucosylated agalactosyl biantennary glycans present on IgG (referred to as the IgG G0 glycoform) which are associated with increasing disease pathogenesis. Protein expression analysis using 2D-DiGE returned a number of differentially expressed protein candidates in the depleted serum, many of which were shown to carry triantennary SLe(X) during subsequent glycomic investigations. Biological pathway analysis of the experimental data returned complement activation and acute phase response signaling as the most significantly altered pathways in the stomach cancer patient serum. Upon the basis of these findings, it is suggested that increased expression of IgG G0 and complement activation are a host response to the presence of the stomach tumor while the increased expression of SLe(X) and acute phase response proteins is a result of pro-inflammatory cytokine signaling, including IL-6, during carcinogenesis. The approach presented herein provides an insight into the underlying mechanisms of disease and the resulting changes in the glycome and glycoproteome offer promise as potential markers for diagnosis and prognostic monitoring in stomach cancer.

Background: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.Results: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. Conclusion:We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.

There is no gold standard assessment tool for TSCs. Heterogeneity in study methods hinders conclusive identification of the most appropriate way to measure TSCs. Subjective measures may reflect the patient experience and more reliably predict changes in dietary behaviour. Evaluation of TSCs should form part of all nutritional assessments in cancer patients. The true prevalence and severity of TSCs at all stages of cancer could then be established.

BackgroundThe glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins.ResultsChinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG.ConclusionsThe use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.