Niaobh O'Donoghue

Variability, heritability and environmental determinants of human plasma N-glycome

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.

Identification of the phosphotyrosine proteome from thrombin activated platelets

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), PI3k (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.

Ultra Performance Liquid Chromatographic Profiling of Serum N-Glycans for Fast and Efficient Identification of Cancer Associated Alterations in Glycosylation

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.

2D-DIGE as a Strategy To Identify Serum Markers for the Progression of Prostate Cancer

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.

In vitro nuclear interactome of the HIV-1 Tat protein

BackgroundOne 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.ResultsOur 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.ConclusionWe 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.

Glycan Characterization of PSA 2-DE Subforms from Serum and Seminal Plasma

Prostate-specific antigen (PSA) two-dimensional electrophoresis (2-DE) subforms (F1-F5) have been described to be altered in prostate cancer (PCa) compared to benign prostatic hyperplasia (BPH). To understand their molecular differences, characterization of these subforms from PCa serum and seminal plasma, namely, at the glycan level, was performed. PSA 2-DE subforms from two serum PCa samples and seminal plasma were analyzed by N-glycan sequencing using high-performance liquid chromatography (HPLC) combined with exoglycosidase array digestions and by mass spectrometry. F1, F2, and F3 subforms showed the same N-glycan pattern, which contained higher levels of sialic acid than the F4 subform, whereas the F5 subform was unglycosylated. When comparing PSA subforms from PCa with seminal plasma, a decrease in sialylation was observed. Furthermore, the analysis of F3, the more abundant PSA subform, showed a higher proportion of alpha 2-3 sialic acid and a decrease in core fucosylated glycans in the PCa sample. These N-glycan changes in PCa PSA subforms highlight the importance of glycosylation as an indicator of PCa disease.

CyDye immunoblotting for proteomics: co-detection of specific immunoreactive and total protein profiles.

The development of ECL‐Plex CyDye‐conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre‐labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2‐D immunoprobing and protein confirmation following MS analysis.

The impact of tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS)

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2‐deficient mice are highly resistant to lipopolysaccharide (LPS)‐induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2−/− macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2‐D DIGE was applied to compare protein patterns from wild‐type and Tyk2−/− macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty‐one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N‐myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS‐induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS‐mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.

Fasting status as a consideration for human serum collection and preparation prior to depletion and analysis

The use of comparative serum proteomic analysis has the potential to reveal protein expression changes present at different stages of disease progression. Depletion strategies allow for the enrichment of low‐abundance proteins, which are more likely to be clinically significant biomarkers. We have observed that patient serum samples filtered through 0.22 μm cellulose acetate spin filters prior to depletion showed a variable level of retention of patient material on the upper part of the filter. This could potentially be related to the fasting status of the patient as a reduction in the lipid content of samples through the incorporation of a centrifugation step prior to filtration was found to reduce this effect. In order to determine if proteins were being selectively retained during filtration, a 2‐D difference gel electrophoresis (2‐D DIGE) experiment was performed. This demonstrated no significant selective retention of protein within crude serum samples. However, as this analysis was carried out on crude serum, it must be emphasised that protein loss could be manifest in the low‐abundance proteins which would be masked in our analysis. Depletion of the retentate was not possible due to technical limitations, however based on our results a centrifugation step might act as an alternative to filtration in serum processing prior to depletion.