Bradley J. Walsh

Complementing genomics with proteomics: The membrane subproteome of Pseudomonas aeruginosa PAO1

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two‐dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane‐spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell‐cell signalling and pathogen‐host interactions in a variety of P. aeruginosa strains.

Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels

Progress in the field of proteomics is dependent upon an ability to visualise close to an entire protein complement via a given array technology. These efforts have previously centred upon two‐dimensional gel electrophoresis in association with immobilised pH gradients in the first dimension. However, limitations in this technology, including the inability to separate hydrophobic, basic, and low copy number proteins have hindered the analysis of complete proteomes. The challenge is now to overcome these limitations through access to new technology and improvements in existing methodologies. Proteomics can no longer be equated with a single two‐dimensional electrophoresis gel. Greater information can be obtained using targeted biological approaches based upon sample prefractionation into specific cellular compartments to determine protein location, while novel immobilised pH gradients spanning single pH units can be used to display poorly abundant proteins due to their increased resolving power and loading capacity. In this study, we show the effectiveness of a combined use of two differential subproteomes (as defined by relative solubilities, cellular location and narrow‐range immobilised pH gradients) to increase the resolution of proteins contained on two‐dimensional gels. We also present new results confirming that this method is capable of displaying up to a further 45% of a given microbial proteome. Subproteomics, utilising up to 40 two‐dimensional gels per sample will become a powerful tool for near‐to‐total proteome analysis in the postgenome era. Furthermore, this new approach can direct biological focus towards molecules of specific interest within complex cells and thus simplify efforts in discovery‐based proteome research.

Urinary biomarkers involved in type 2 diabetes: a review

Diabetes mellitus is one of the most challenging health concerns of the 21st century. With at least 30% of the diabetic population remaining undiagnosed, effective and early diagnosis is of critical concern. Development of a diagnostic test, more convenient and reliable than those currently used, would therefore be highly beneficial. Urine as a diagnostic medium allows for non‐invasive detection of biomarkers, including some associated with type 2 diabetes and its complications. This review provides a synopsis of those urinary biomarkers that potentially may provide a basis for the development of improved diagnostic tests. Three main pathways for the sourcing of potential makers are identified: kidney damage, oxidative stress and low‐grade inflammation including atherosclerosis/vascular damage. This review briefly presents each pathway and some of the most relevant urinary biomarkers that may be used to monitor the development or progression of diabetes and its complications. In particular, biomarkers of renal dysfunction such as transferrin, type IV collagen and N‐acetyl‐β‐D‐glucosaminidase might prove to be more sensitive than urinary albumin, the current gold standard, in the detection of incipient nephropathy and risk assessment of cardiovascular disease. Inflammatory markers including orosomucoid, tumour necrosis factor‐α, transforming growth factor‐β, vascular endothelial growth factor and monocyte chemoattractant protein‐1, as well as oxidative stress markers such as 8‐hydroxy‐2′deoxyguanosine may also be useful biomarkers for diagnosis or monitoring of diabetic complications, particularly kidney disease. However, the sensitivity of these markers compared with albumin requires further investigation. Copyright

Microplate reader-based quantitation of collagens

By using a picrosirius dye, sensitive and specific staining of collagens plated in microtiter wells was achieved. The range of detection was from 0.5 to 20 micrograms. Human collagen types I, III, IV, and V were tested and able to be detected by the method. The dye did not bind to acetylcholinesterase or elastin. It did bind to C1q to some extent but this is not surprising since the molecule contains some triple helical collagen-like structures. A comparison performed between this assay and a colorimetric assay for hydroxyproline using tissue culture supernatants gave similar results for both samples. Due to its simplicity and sensitivity this assay will be most useful in laboratories where large numbers of samples must be screened for collagen production.

Comparative proteomics of bacterial pathogens.

The monitoring of gene expression via the technologies encompassed under the term ‘proteomics’ allows proteins of significance to be related to phenotypes associated with strain variability, environmental influences and the effects of genetic manipulation. The characterisations of these molecules are routinely performed utilising two‐dimensional (2‐D) gel electrophoresis in association with mass spectrometry for the identification of proteins. Pathogenic bacteria are suitable for proteomic comparisons in the aim of elucidating proteins with vaccine and diagnostic applications, as well as determining novel targets for drug design and the effects of these drugs on cellular physiology. Strains exhibiting diverse phenotypes including antibiotic or chemical resistances, altered mode of pathogencity, or differential capability of growth in similar environments, can be compared via protein differential display to correlate relative protein abundances associated with these conditions. Technically, proteins are ‘mapped’ on 2‐D arrays under ‘standard’ conditions and visually compared to arrays of proteins from a variety of test conditions. High‐throughput technologies allow molecules of significance to be elucidated rapidly from within complex mixtures using a combination of cellular pre fractionation to determine cellular location and pathway predictions to aid in overcoming the limitations of 2‐D gel technology for the analysis of whole proteomes.

Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa can be phenotypically classified by their mode of pathogenicity as either invasive, where the bacterium is internalised by host cells, or cytotoxic, where the host cell is killed without internalisation through the expression of cytotoxicity factors. These phenotypes are thought to depend primarily on the interactions of pseudomonal membrane and secreted proteins with host cells. We report here comparisons of outer membrane and extracellular protein‐enriched fractions from invasive (PAO1) and cytotoxic (6206) strains of P. aeruginosa separated by two‐dimensional (2‐D) gel electrophoresis. Gel image comparisons revealed the two strains express essentially identical membrane protein profiles under the conditions investigated. Membrane protein strain differences were typically the result of minor amino acid sequence variations resulting in small mass and isoelectric point shifts visible on 2‐D gels. Analysis of extracellular proteins from stationary phase growth, however, revealed significantly different protein profiles. Extracellular fractions from the invasive PAO1 strain were dominated by extracellular proteases including elastase (LasB), LasA protease and chitin‐binding protein, as well as several previously designated ‘hypothetical’ proteins. LasB appeared to be highly processed with 28 discrete mass and isoelectric point forms detected in this study. The significant number of active extracellular proteases (including LasB itself) may account for this processing. Conversely, extracellular fractions from strain 6206 consisted mainly of cellular and membrane exposed proteins including GroEL, DnaK and flagellar subunits. These are thought to result from cellular turnover during growth and the reliance on the secretory mechanisms of this strain to produce high levels of cytotoxicity factors, such as ExoU, which may be produced only upon specific interactions with host cells. These studies will aid in elucidating the differences between invasive and cytotoxic P. aeruginosa at the proteome level.

A method for the detection of IgE binding sequences of allergens based on a modification of epitope mapping

An ELISA method for the rapid determination of IgE binding sites (allergenic determinants) of proteins is reported. The method utilizes the epitope mapping kit (Geysen et al., 1984) to synthesize hexapeptides of an allergen of interest, followed by a biotin-avidin system to detect peptide-bound IgE. The technique allows rapid localisation of determinants from allergens of known sequence without the need to purify large amounts of allergen nor to generate peptides by cleavage of it. Using the results of the epitope mapping experiments a putative allergenic peptide containing 18 amino acid residues from the sequence of a wheat allergen was identified and synthesised on polyamide resin. Testing of this peptide by radioallergosorbent test (RAST) inhibition showed that it bound specific IgE in the sera of patients allergic to wheat.

Interaction of Immune and Connective Tissue Cells: I. The Effect of Lymphokines and Monokines on Fibroblast Growth

In order to determine if mononuclear cells may be secreting factors capable of modulating fibroblast growth, the in vitro proliferative response of fibroblasts to cytokines known to be secreted by mononuclear cells was measured, using both growth arrested and proliferating cells. Of the cytokines tested, which included interleukin‐1 (IL‐1), interleukin‐2 (IL‐2), interleukin‐3 (IL‐3), interleukin‐4 (IL‐4), interleukin‐6 (IL‐6), interferon‐alpha (IFN‐alpha), interferon‐gamma (IFN‐gamma), transforming growth factor‐alpha (TGF‐alpha), transforming growth factor‐beta (TGF‐beta), platelet derived growth factor (PDGF), and tumor necrosis factor‐alpha (TNF‐alpha), only TNF‐alpha and PDGF had demonstrable growth factor activity. Neither IL‐1 alpha nor beta showed any true growth factor activity but were able to enhance the replication of already proliferating cells. No inhibition of proliferation was noted by any of the cytokines with the exception of TNF‐alpha and TGF‐beta. TNF‐alpha in doses greater than 500 ng/ml caused fibroblast death, probably by a prostaglandin related mechanism as fibroblasts remained viable, although non proliferative, when assayed in the presence of indomethacin, a known inhibitor of prostaglandin E2 (PGE2) synthesis. TGF‐beta was inhibitory to proliferation at doses greater than 100 ng/ml, while fibroblasts remained viable. This effect was not influenced by indomethacin and hence is unlikely to be PGE2 related.

Innovative biomarkers for prostate cancer early diagnosis and progression

The marker currently used for prostate cancer (CaP) detection is an increase in serum prostate-specific antigen (PSA). However, the PSA test which may give false positive or negative information, is not reliable and does not allow the differentiation of benign prostate hyperplasia (BPH), non-aggressive CaP and aggressive CaP. There is thus an urgent need to search for novel CaP biomarkers to improve the early detection and accuracy of diagnosis, determine the aggressiveness of CaP and to monitor the efficacy of treatment. Proteomic techniques allow for a high-throughput analysis of bio-fluids with the visualization and quantification of thousands of potential protein markers and represent very promising tools in the search for new, improved molecular markers of CaP. In this review, we will summarize conventional CaP biomarkers and focus on novel identified biomarkers for CaP early diagnosis and progression that might be used in the future.

Lacryglobin in human tears, a potential marker for cancer.

Lacryglobin has been identified in human tears. This protein has high sequence homology to the mammaglobins, proteins upregulated in breast cancer and in breast cancer metastasis. In order to investigate the utility of tear screening for cancer, tear samples were collected from patients with different types of cancer and compared to controls. Tear samples were taken from five controls and eight breast, six lung, five colon, one prostate and three ovary cancer patients. Tears were analysed using 2‐D gel electrophoresis (n = 25) and 1‐D electrophoresis (n = 3). Lacryglobin was present in the following percentage of patients: breast cancer (88%), lung (83%), colon (100%), ovary (33%), prostate (100%) and controls (60%). Two control patients with lacryglobin had a family history of breast and prostate cancer. Lacryglobin was detected in some but not all tear samples and further studies are warranted to investigate its potential as a marker for cancer.