Richard Lipscombe

Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion)

Venoms have evolved over millions of years into potent cocktails of bioactive peptides and proteins. These compounds can be of great value to the pharmaceutical industry for numerous clinical applications. In this study, a novel proteomic – bioinformatic approach was utilised, where chromatography followed by gel electrophoresis was utilised to separate the venom peptides/proteins of Heterometrus longimanus (Asian black scorpion). Purified peptides were analysed by tandem mass spectrometry, de novo sequenced and then homology matched against known peptides in the Swiss‐Prot protein database. Numerous potentially biologically active peptide matches were discovered, and a simple scoring system applied to putatively assign functions to the peptides. As a validation of this approach, the functional composition of the experimentally derived proteome is similar to that of other scorpions, and contains a potent mix of toxins, antimicrobials and ionic channel inhibitors.

Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum

BackgroundStagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained.ResultsIn this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellular-associated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome.ConclusionWe conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies.

A functional genomics approach to dissect the mode of action of the Stagonospora nodorum effector protein SnToxA in wheat

In this study, proteomics and metabolomics were used to study the wheat response to exposure to the SnToxA effector protein secreted by the fungal pathogen Stagonospora nodorum during infection. Ninety-one different acidic and basic proteins and 101 metabolites were differentially abundant when comparing SnToxA- and control-treated wheat leaves during a 72-h time course. Proteins involved in photosynthesis were observed to increase marginally initially after exposure, before decreasing rapidly and significantly. Proteins and metabolites associated with the detoxification of reactive oxygen species in the chloroplast were also differentially abundant during SnToxA exposure, implying that the disruption of photosynthesis causes the rapid accumulation of chloroplastic reactive oxygen species. Metabolite profiling revealed major metabolic perturbations in central carbon metabolism, evidenced by significant increases in tricarboxylic acid (TCA) cycle intermediates, suggestive of an attempt by the plant to generate ATP and reducing equivalents in response to the collapse of photosynthesis caused by SnToxA. This was supported by the observation that the TCA cycle enzyme malate dehydrogenase was up-regulated in response to SnToxA. The infiltration of SnToxA also resulted in a significant increase in abundance of many pathogenicity-related proteins, even in the absence of the pathogen or other pathogen-associated molecular patterns. This approach highlights the complementary nature of proteomics and metabolomics in studying effector-host interactions, and provides further support for the hypothesis that necrotrophic pathogens, such as S. nodorum, appear to exploit existing host cell death mechanisms to promote pathogen growth and cause disease.

Evidence of Altered Guinea Pig Ventricular Cardiomyocyte Protein Expression and Growth in Response to a 5 min in vitro Exposure to H2O2

Oxidative stress and alterations in cellular calcium homeostasis are associated with the development of cardiac hypertrophy. However, the early cellular mechanisms for the development of hypertrophy are not well understood. Guinea pig ventricular myocytes were exposed to 30 microM H(2)O(2) for 5 min followed by 10 units/mL catalase to degrade the H(2)O(2), and effects on protein expression were examined 48 h later. Transient exposure to H(2)O(2) increased the level of protein synthesis more than 2-fold, assessed as incorporation of [(3)H]leucine (n = 12; p < 0.05). Cell size was increased slightly, but there was no evidence of major cytoskeletal disorganization assessed using fluorescence microscopy. Changes in the expression of individual proteins were assessed using iTRAQ protein labeling followed by mass spectrometry analysis (LC-MALDI-MSMS); 669 proteins were identified, and transient exposure of myocytes to H(2)O(2) altered expression of 35 proteins that were predominantly mitochondrial in origin, including TCA cycle enzymes and oxidative phosphorylation proteins. Consistent with changes in the expression of mitochondrial proteins, transient exposure of myocytes to H(2)O(2) increased the magnitude of the mitochondrial NADH signal 10.5 +/- 2.3% compared to cells exposed to 0 microM H(2)O(2) for 5 min followed by 10 units/mL catalase (n = 8; p < 0.05). In addition, metabolic activity was significantly increased in the myocytes 48 h after transient exposure to H(2)O(2), assessed as formation of formazan from tetrazolium salt. We conclude that a 5 min exposure of ventricular myocytes to 30 microM H(2)O(2) is sufficient to significantly alter protein expression, consistent with the development of hypertrophy in the myocytes. Changes in mitochondrial protein expression and function appear to be early sequelae in the development of hypertrophy.

Detecting changes in the thiol redox state of proteins following a decrease in oxygen concentration using a dual labeling technique.

Cells are routinely exposed to hyperoxic conditions when cultured in the presence of 95% air and 5% carbon dioxide. Hyperoxic conditions can increase the generation of reactive oxygen species and cause oxidative stress. Oxidative stress has been proposed to cause cells in culture to behave differently from cells in vivo. One route by which oxidative stress could affect cellular function is through alterations in protein function caused by the oxidation of thiol groups (-SH) of redox-sensitive cysteine residues. To test whether changes in oxygen concentration were sufficient to cause changes in the thiol redox state of proteins, we developed a sensitive method involving the labeling of reduced and oxidized cysteine residues with fluorescent tags. Using this dual labeling method, we found 62 of 411 protein spots that were significantly more reduced following a 30 min decrease in oxygen concentration. We conclude that the elevated oxygen concentration characteristic of typical cell culture conditions has the potential to affect cellular behavior through changes in the thiol redox state of proteins.

Quantitative proteomic analysis of G-protein signalling in Stagonospora nodorum using isobaric tags for relative and absolute quantification.

The G protein α‐subunit (Gna1) in the wheat pathogen Stagonospora nodorum has previously been shown to be a critical controlling element in disease ontogeny. In this study, iTRAQ and 2‐D LC MALDI‐MS/MS have been used to characterise protein expression changes in the S. nodorum gna1 strain versus the SN15 wild‐type. A total of 1336 proteins were identified. The abundance of 49 proteins was significantly altered in the gna1 strain compared with the wild‐type. Gna1 was identified as having a significant regulatory role on primary metabolic pathways, particularly those concerned with NADPH synthesis or consumption. Mannitol dehydrogenase was up‐regulated in the gna1 strain while mannitol 1‐phosphate dehydrogenase was down‐regulated providing direct evidence of Gna1 regulation over this enigmatic pathway. Enzymatic analysis and growth assays confirmed this regulatory role. Several novel hypothetical proteins previously associated with stress and pathogen responses were identified as positively regulated by Gna1. A short‐chain dehydrogenase (Sch3) was also significantly less abundant in the gna1 strains. Sch3 was further characterised by gene disruption in S. nodorum by homologous recombination. Functional characterisation of the sch3 strains revealed their inability to sporulate in planta providing a further link to Gna1 signalling and asexual reproduction. These data add significantly to the identification of the regulatory targets of Gna1 signalling in S. nodorum and have demonstrated the utility of iTRAQ in dissecting signal transduction pathways.

Contribution of the membrane-distal tyrosine in intracellular signaling by the granulocyte colony-stimulating factor receptor

We have evaluated the contribution of intracellular tyrosine residues of the granulocyte colony-stimulating factor receptor (GCSF-R) to its signaling and cellular outcomes. We began with stable BaF3 cell lines overexpressing wild-type or mutant GCSF-Rs. When all four intracellular tyrosines of the GCSF-R were replaced with phenylalanine (FFFF GCSF-R), cell proliferation and survival were compromised. Replacement of only the membrane-distal tyrosine (YYYF GCSF-R) also showed reduced survival following a GCSF withdrawal/replacement protocol, suggesting a role for this tyrosine. Proliferation by FFFY GCSF-R cells was attenuated by ∼70%. In evaluating the biochemical steps involved in signaling, we then showed that the membrane-distal tyrosine was necessary and sufficient for c-Jun N-terminal kinase (JNK) activation. With the use of a cell-permeable JNK-inhibitory peptide, JNK was implicated in the proliferation of the FFFY GCSF-R mutant. To further define the events linking the membranedistal tyrosine and JNK activation, the Src homology 2 domains of Shc, Grb2, and 3BP2 were shown to bind the full-length GCSF-R and a phosphopeptide encompassing the membrane-distal tyrosine. When binding to variant phosphopeptides based on this membrane-distal tyrosine was tested, altering the amino acids immediately following the phosphotyrosine could selectively abolish the interaction with Shc or Grb2, or the binding to both Grb2 and 3BP2. When these changes were introduced into the full-length GCSF-R and new cell lines created, only the mutant that did not interact with Grb2 and 3BP2 did not activate JNK. Our results suggest that direct binding of Shc by the GCSF-R is not essential for JNK activation.

Expression of cardiac α-actin spares extraocular muscles in skeletal muscle α-actin diseases – Quantification of striated α-actins by MRM-mass spectrometry

As with many skeletal muscle diseases, the extraocular muscles (EOMs) are spared in skeletal muscle alpha-actin diseases, with no ophthalmoplegia even in severely affected patients. We hypothesised that the extraocular muscles sparing in these patients was due to significant expression of cardiac alpha-actin, the alpha-actin isoform expressed in heart and foetal skeletal muscle. We have shown by immunochemistry, Western blotting and a novel MRM-mass spectrometry technique, comparable levels of cardiac alpha-actin in the extraocular muscles of human, pig and sheep to those in the heart. The sparing of extraocular muscles in skeletal muscle alpha-actin disease is thus probably due to greater levels of cardiac alpha-actin, than the negligible amounts in skeletal muscles, diluting out the effects of the mutant skeletal muscle alpha-actin.

Identification of Novel Circulating Biomarkers Predicting Rapid Decline in Renal Function in Type 2 Diabetes: The Fremantle Diabetes Study Phase II

OBJECTIVE To assess the ability of plasma apolipoprotein (apo) A-IV (apoA4), apo C-III, CD5 antigen-like (CD5L), complement C1q subcomponent subunit B (C1QB), complement factor H–related protein 2, and insulin-like growth factor binding protein 3 (IBP3) to predict rapid decline in estimated glomerular filtration rate (eGFR) in type 2 diabetes. RESEARCH DESIGN AND METHODS Mass spectrometry was used to measure baseline biomarkers in 345 community-based patients (mean age 67.0 years, 51.9% males) from the Fremantle Diabetes Study Phase II (FDS2). Multiple logistic regression was used to determine clinical predictors of rapid eGFR decline trajectory defined by semiparametric group-based modeling over a 4-year follow-up period. The incremental benefit of each biomarker was then assessed. Similar analyses were performed for a ≥30% eGFR fall, incident chronic kidney disease (eGFR <60 mL/min/1.73 m2), and eGFR decline of ≥5 mL/min/1.73 m2/year. RESULTS Based on eGFR trajectory analysis, 35 participants (10.1%) were defined as “rapid decliners” (mean decrease 2.9 mL/min/1.73 m2/year). After adjustment for clinical predictors, apoA4, CD5L, and C1QB independently predicted rapid decline (odds ratio 2.40 [95% CI 1.24–4.61], 0.52 [0.29–0.93], and 2.41 [1.14–5.11], respectively) and improved model performance and fit (P < 0.001), discrimination (area under the curve 0.75–0.82, P = 0.039), and reclassification (net reclassification index 0.76 [0.63–0.89]; integrated discrimination improvement 6.3% [2.1–10.4%]). These biomarkers and IBP3 contributed to improved model performance in predicting other indices of rapid eGFR decline. CONCLUSIONS The current study has identified novel plasma biomarkers (apoA4, CD5L, C1QB, and IBP3) that may improve the prediction of rapid decline in renal function independently of recognized clinical risk factors in type 2 diabetes.

Comprehensive mass spectrometry based biomarker discovery and validation platform as applied to diabetic kidney disease

Highlights • A direct proteomics-based biomarker discovery to validation workflow is proposed.• Targeted mass spectrometry enabled robust multiplexing assays.• The mass spectrometry assay demonstrated CVs of: intra-day 5.9% and inter-day 8.1%.• A protein biomarker panel has been developed specific for diabetic kidney disease.• The biomarker panel presented outperforms current gold standard tests.