Search

The term "proteome" was first introduced into the scientific literature in July 1995. Almost 20 years ago attempts to characterize the "total protein complement able to be encoded by a given genome" only became possible due to privileged access to what were then the world's most complete sets of genomic data. Today, proteomics has become an important pillar in the fields of disease diagnosis and drug research and development, while also playing a critical role in the much larger field of Healthcare Analytics and Biomarker Discovery and Detection. It is important to note that this industry originated mostly from building blocks in analytical science that predated the term "proteomics" by many decades. However, proteomics, as a discipline, has allowed protein scientists to more favorably compete in the face of highly fashionable Big Science and, more specifically, genomics.

Bacteriophages have a potentially important role to play in reducing the global incidence of Hospital Acquired Infection (HAI). Their use should be focused on reducing the use and over-use of antibiotics as part of integrated control measures in conjunction with various vaccination, sanitation procedures and prophylactic and treatment regimens. Bacteriophages offer exquisite specificity and efficacy in killing target bacterial strains, a phenomenon known for almost 100 years. However, their efficacy with respect to broad-spectrum antibiotics is poor due to the highly strain-selective nature of their killing and their rapid elimination from the body. Bacteriophage killing is a naturally-occurring process capable of limiting and eliminating bacterial populations in humans. This is achieved through exponential amplification of their number, if and when, they encounter a target bacterium. Unfortunately, processes employed for their commercial production today do not meet the same rigour as dictated for pharmaceutical products. Batch-to-batch reproducibility and molecular definition of target and phage strains must be demanded before their clinical use can become widespread. Elsewhere, historical data have demonstrated safety in humans beyond any doubt. Because patients continue to die in our healthcare centers internationally, the use of bacteriophage to help fight HAI should be reassessed. Here, relevant literature is reviewed.

A mouse model for nonatopic asthma was employed to study the alterations of the lung proteome to gain insight into the underlying molecular mechanisms of disease pathophysiology post-challenge. Lung samples from asthmatic and control mice were used to generate 24 high quality two-dimensional electrophoresis gels wherein 2115 proteins were examined for disease relevance. In total, 23 proteins were significantly up- or down-regulated following hapten-challenge of dinitro-fluorobenzene-hypersensitive mice. Twenty proteins were identified by mass spectrometry, of which 18 could be linked to asthma related symptoms, such as stress and inflammation, lung detoxification, plasma exudation and/or tissue remodeling. As such, proteomics was clearly vindicated as a means of studying this complex disease phenomenon. The proteins found in this study may not necessarily play a role in the immunological mechanisms and/or pathophysiology of asthma development. However, they may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy. The mathematics of achieving statistical confidence from low numbers of gel replicates containing large numbers of independent variables stress the need for high numbers of replicates to better sample the population of proteins revealed by two-dimensional gel electrophoresis.

Mycoplasma genitalium is the smallest member of the class Mollicutes, with a genome size of 580 kb. It has the potential to express 480 gene products, and is therefore considered to be an excellent model to assess: (a) the minimum metabolism required by a free living cell; and (b) proteomic technologies and the information obtained by proteome analysis. Here, we report on the most complete proteome observed at 73% (expected proteome), and analysed at 33% (reported proteome). The use of four overlapping pH windows in conjunction with SDS/PAGE has allowed 427 distinct proteins to be resolved in association with the exponential growth of M. genitalium. Proof of expression for 201 proteins of sufficient abundance on silver stained two-dimensional gels was obtained using peptide mass fingerprinting (PMF) of which 158 were identified. The potential for gene product modification in even the simplest known self-replicating organism was quantified at a ratio of 1.22 : 1, more proteins than genes. A reduction in protein expression of 42% was observed for post-exponentially-grown cells. DnaK, GroEL, DNA gyrase, and a cytadherence accessory protein were significantly elevated, while some ribosomal proteins were reduced in relative abundance. The strengths and weaknesses of techniques employed were assessed with respect to the observed and predicted proteome derived from DNA sequence information. Proteomics was shown to provide a perspective into the biochemical and metabolic activities of this organism, beyond that obtainable by sequencing alone.

The identity of 45 protein spots representing 32 orthologues within the Ochrobactrum anthropi proteome within a gradient of pH 4-7, and mass range 5-90 kDa were determined across species boundaries. These proteins could be classified into 13 functional categories and establish metabolic, regulatory and translatory systems including amino acid biosynthesis, electron transport and the potential for plant symbiosis in a molecularly understudied organism. Amino acid composition and/or peptide mass fingerprinting were employed as a means to search the Swiss-Prot and OWL protein sequence databases for similarity within a broad taxonomic class of bacteria. Candidate matches from database searches could be compared and a simple multiplication matrix based on co-occurrence and rank within the top 96 most similar entries was used to provide statistical confidence. This mathematical matrix was evaluated with respect to the characterisation of O. anthropi, an unsequenced and understudied bacterium, in the light of the recent influx of DNA sequence information.

Replication-induced protein synthesis (RIPS) can occur following the passage of the replisome due to transcription initiated by RNA polymerase in association with: (i) negative supercoiling trailing the replisome / replication fork, (ii) hemimethylation prior to the action of dam methylase, (iii) transient derepression following passage of the replisome / replication fork and prior to renewed synthesis of the repressor gene-product, and (iv) “sliding clamp” accessory DNA-binding proteins binding to the lagging strand DNA duplex to retard rotational upstream propagation of supercoils. The latter include subunits of DNA polymerase III in Escherichia coli and gp45 in T4 bacteriophage. By far the most convincing evidence for the existence of RIPS comes from the pulse of protein synthesis which follows the passage of the replisome in late T4 bacteriophage, the dynamics of replication in Escherichia coli, recent results from cDNA high-density expression arrays in yeast and the workings of the lac-operon. More circumstantial evidence is provided by “leaky” or “aberrant” protein expression in genetic systems where attempts have been made to turn off protein synthesis by molecular means. In higher vertebrates, RIPS may have a potentially important role in explaining the mechanisms by which thymic and peripheral immune self-tolerance is established, either directly through antigen presentation on dendritic cells or through the presentation of peptides derived from T-cells. The latter model is preferred, as young T-cells will have recently divided and will be dying in large numbers near the antigen-presenting dendritic cells in the thymus. The functional utility of RIPS would appear to be linked to both facilitating cellular metabolism and an improved survival during stress. RIPS, as a potentially universal molecular phenomenon, presents proteomics with numerous challenges and opportunities, both technical and commercial.

The EcoGene project involves the examination of Escherichia coli K-12 DNA sequences and accompanying annotation in the public databases in order to refine the representation and prediction of the entire set of E. coli K-12 chromosomally encoded protein sequences. The results of this ongoing effort have been deposited in the SWISSPROT protein sequence database as sequencing of the E. coli genome has progressed to completion in recent years. Through this continuing research, we have discovered that the prediction of low molecular weight (small) proteins, arbitrarily defined as protein sequences < or = 150 amino acids (aa) in length, is problematic and requires special attention. We describe the small protein subset of EcoGene and the approach used to derive this subset from the complete E. coli genome sequence and database annotations. These E. coli proteins have helped to identify new small genes in other organisms and to identify conserved residues (motifs) using database searches and multiple alignments. Two thirds of the E. coli small proteins have not been characterized experimentally. The careful application of computer and laboratory methods to the analysis of small proteins is needed for accurate prediction, verification and characterization. The problem of accurate protein sequence identification is not limited to small proteins or to E. coli; these problems are encountered to varying degrees throughout all sequence databases.

The malate (MDH) and lactate (LDH) dehydrogenases belong to the homologous class of 2-ketoacid dehydrogenases. The specificity for their respective substrates depends on residues differing at two or three regions within each molecule. Theoretical peptide-mass fingerprinting and PROSITE analysis of nine MDH and six LDH molecules were used to describe conserved sites related to function. A unique LDH is described which probably also confers MDH activity within the 580 kbp genome of Mycoplasma genitalium (class: Mollicutes). A single hydrophilic arginine residue was found in the active site of the M. genitalium LDH enzyme, differing from an hydrophobic residue normally present in these molecules. The effect of this residue may be to alter active site substrate specificity, allowing the enzyme to perform two closely related tasks. Evidence for a single gene affording dual enzymatic function is discussed in terms of genome size reduction in the simplest of free-living organisms. Since Mollicutes are thought to lack enzymes of the tricarboxylic acid cycle that would otherwise bind and interact with MDH in bacterial species possessing this pathway, active site modification of M. genitalium LDH is the sole requirement for MDH activity of this molecule. The closely related helical Mollicute, Spiroplasma melliferum, was shown to possess two distinct gene products for MDH/LDH activity.

A knowledge of the 'proteome,' total protein output encoded by a genome, provides information on (1) if and when predicted gene products are translated, (2) the relative concentrations of gene products, and (3) the extent of posttranslational modification, none of which can be accurately predicted from the nucleic acid sequence alone. The current status of proteome analysis is reviewed with respect to some of the techniques employed, automation, relevance to genomic studies, mass spectrometry and bioinformatics, limitations, and recent improvements in resolution and sensitivity for the detection of protein expression in whole cells, tissues, or organisms. The concept of 'proteomic contigs' is introduced for the first time. Traditional approaches to genomic analysis call upon a number of strategies to produce contiguous DNA sequence information, while 'proteomic contigs' are derived from multiple molecular mass and isoelectric point windows in order to construct a picture of the total protein expression within living cells. In higher eukaryotes, the latter may require several dozen image subsets of protein spots to be stitched together using advanced image analysis. The utility of both experimental and theoretical peptide-mass fingerprinting (PMF) and associated bioinformatics is outlined. A previously unknown motif within the peptide sequence of Elongation Factor Tu from Thermus aquaticus was discovered using PMF. This motif was shown to possess potential significance in maintaining structural integrity of the entire molecule.

Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined. Theoretical and experimental data were used to demonstrate that peptide-mass fingerprinting (PMF) could aid in the recognition of conserved motifs across species boundaries, and thereby assist in attributing putative function to some of these molecules. Amino acids residue substitutions produced by biological diversity and phylogenetic distance combine to highlight regions of functional significance within proteins. Using 10 prokaryotic and two eukaryotic elongation factors (EF), up to 25 peptide fragments (> 800 Da) per molecule were compared across species boundaries within a 12 x 12 contingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity. Total amino acid sequence identity ranged from 29.4-80.9% for these molecules. Peptide fragments with homologous sequence across three or more EF were defined as containing, or being near to, conserved functional motifs. Twelve such fragments (> 800 Da) were found in this group of proteins. In addition, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules. At the 83% (five of six residues) identity level, this fragment was found in a further 35 EF-Tu molecules and in 14 unrelated proteins. Further investigation should reveal a role for this fragment (motif) in structural integrity or protein function. A FASTA search conducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to putatively attribute partial function to three hypothetical proteins derived from DNA sequencing initiatives.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3-10 and narrow range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N-terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI approximately 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6-11) IPGs has allowed the visualisation of a significantly greater percentage of the 'functional proteome', that portion of the total protein complement of a genome actively translated within a specific time frame, on 2-D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.

The rules that govern the dynamics of protein characterisation by peptide-mass fingerprinting (PMF) were investigated through multiple interrogations of a nonredundant protein database. This was achieved by analysing the efficiency of identifying each entry in the entire database via perfect in silico digestion with a series of 20 pseudo-endoproteinases cutting at the carboxy terminal of each amino acid residue, and the multiple cutters: trypsin, chymotrypsin and Glu-C. The distribution of peptide fragment masses generated by endoproteinase digestion was examined with a view to designing better approaches to protein characterisation by PMF. On average, and for both common and rare cutters, the combination of approximately two fragments was sufficient to identify most database entries. However, the rare cutters left more entries unidentified in the database. Total coverage of the entire database could not be achieved with one enzymatic cutter alone, nor when all 23 cutters were used together. Peptide fragments of > 5000 Da had little effect on the outcome of PMF to correctly characterise database entries, while those with low mass (near to 350 Da in the case of trypsin) were found to be of most utility. The most frequently occurring fragments were also found in this lower mass region. The maximum size of uncut database entries (those not containing a specific amino acid residue) ranged from 52,908 Da to 258,314 Da, while the failure rate for a single cutter in identifying database entries varied from 10,865 (8.4%) to 23,290 (18.1%). PMF is likely to be a mainstay of any high-throughput protein screening strategy for large-scale proteome analysis. A better understanding of the merits and limitations of this technique will allow researchers to optimise their protein characterisation procedures.

Spiroplasma melliferum (Class: Mollicutes) is a wall-less, helical bacterium with a genome of approximately 1460 kbp encoding 800-1000 gene-products. A two-dimensional electrophoresis gel reference map of S. melliferum was produced by Phoretix 2-D gel software analysis of eight high quality gels. The reference map showed 456 silver-stained and replicated protein spots. 156 proteins (34% of visible protein spots) from S. melliferum were further characterised by one, or a combination, of the following: amino acid analysis, peptide-mass fingerprinting via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry, and N-terminal protein microsequencing. Proteins with close relationship to those previously determined from other species were identified across species barriers. Thus, this study represents the first larger-scale analysis of a proteome based upon the attribution of predominantly 'unique numerical parameters' for protein characterisation across species boundaries, as opposed to a sequence-based approach. This approach allowed all database entries to be screened for homology, as is currently the case for studies based on nucleic acid or protein sequence information. Several proteins studied from this organism were identified as hypothetical, or having no close homolog already present in the databases. Gene-products from major families such as glycolysis, translation, transcription, cellular processes, energy metabolism and protein synthesis were identified. Several gene-products characterised in S. melliferum were not previously found in studies of the entire Mycoplasma genitalium and Mycoplasma pneumoniae (both closely related Mollicutes) genomes. The presence of such gene-products in S. melliferum is discussed in terms of genome size as compared with the smallest known free-living organisms. Finally, the levels of expression of S. melliferum gene-products were determined with respect to total optical intensity associated with all visible proteins expressed in exponentially grown cells.

Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.

Knowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one- and two-dimensional (2-DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of approximately 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4 degrees C) of cell-(CEM-T4, THP-1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS-PAGE immunoblotting, using anti-CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full-length native CD4 from lymphoid MT2 and CEM-T4 cell extracts, however, revealed both monomeric and higher-order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation, 1-DE and 2-DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.

A variety of electrophoretic techniques were used to search for potential causes of human dilated cardiomyopathy (DCM). Northern blots were used to quantify alpha-cardiac and alpha-skeletal muscle actins, and beta-myosin heavy chain mRNAs which are the predominant expressed isoform species. We found a wide range of mRNA levels expressed in both DCM and nondiseased (ND) samples of left ventricles. However, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of the same heart samples revealed a stable and constant ratio of actin and myosin. Dystrophin deficiency might account for the DCM symptoms and so dystrophin levels of DCM and ND samples were evaluated using Western blots probed with monoclonal antibodies for the N-, C- and mid-rod portions of this protein. We found that dystrophin levels were constant in all 29 DCM and 5 ND samples suggesting that dystrophin deficiency is probably not a contributing cause. We explored the possibility that terminal failure may be due to an apoptotic-like event in the cardiomyocytes. Zymograms of DCM and ND samples revealed a significant increase in DNase I activity in the DCM group compared to the ND samples. These data raise the possibility that end-stage failure may be associated with apoptosis.

Histopathology visible in premortem female Anopheles stephensi mosquitoes was examined using light and transmission electron microscopy at 5 days postintrathoracic inoculation with Spiroplasma taiwanense strain SP2/sl/mut/P9. Light microscopy showed thoracic flight muscle to have undergone extensive degradation and polysaccharide depletion. Transmission electron microscopy revealed spiroplasmas both extra- and intra-cellularly and occurring in hemolymph, thoracic flight muscle, glial cells, neural lamella, hemocytes, an unknown cell type found in juxtaposition with axon bundles, connective tissue surrounding diverticulum and midgut, trophocytes, and tracheocytes. Replication of spiroplasmas within cells caused cellular lysis and the liberation of spiroplasmas into the hemolymph. Necrosis of infected muscle cells involved, successively, condensation, swelling, and lysis of mitochondria, Z-disc loss, and splitting of myofibrils. Nerve cord axons surrounded by infected glial cells were distended by swollen mitochondria. Spiroplasmas were able to survive and replicate in hemolymph and within phagocytic vacuoles of hemocytes, independent of any host immune response. Pathologies observed within thoracic flight muscle resulted from intracellular replication of spiroplasmas and probably produced the loss of flight capacity observed for these mosquitoes. Neurological involvement and/or the presence of toxic metabolites may represent additional pathogenic determinants. The existence of life-history stages described previously in vitro were confirmed in vivo .

Amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two-dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS-PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer-generated rankings. Results were verified using N-terminal protein microsequencing. The combined use of amino acid composition and MALDI-TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4-10 tryptic digest fragments). A further two parameters, estimated M(r) and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.

Spiroplasma taiwanense is the first member of the Class Mollicutes to be subjected to polypeptide cartography using computerized image analysis. The small genome size characteristic of this group was shown to code for low numbers of polypeptides when compared to other bacterial species. Silver-stained two-dimensional electrophoresis gels, following separation by either isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (ISO-DALT) or nonequilibrium pH gradient electrophoresis (NEPHGE), were used to create databases from 10 and 6 gels, respectively, for each technique and produced, respectively, 263 and 287 replicated spots. Polypeptides were mapped with respect to molecular mass and glyceraldehyde-3-phosphate dehydrogenase carbamylation standards. Of interest was the unexpectedly high percentage (50.2%) of the total normalised optical intensity associated with all 263 spots detected by ISO-DALT electrophoresis, having been contributed by just 29 dominant protein spots. These 29 polypeptides are to be given priority in microsequencing and microanalysis aimed at their identification.

A tissue-specific variation of a total of 152 mitochondria-associated polypeptides is detailed for etiolated leaves, epicotyls and roots from the pea, Pisum satisum L., using computerized image analysis. These organ systems possess, respectively, 128, 100, and 96 mitochondria-associated polypeptides with 38, 8, and 10, respectively, being unique to each. Seventy-one polypeptides were observed to be present in all gels, but some of these also underwent a variety of quantifiable changes. Two polypeptides were identified that varied similarly to a polypeptide of interest (W), known to be abundant in pea epicotyls and potato tubers. It is postulated that these latter three polypeptides are therefore probably related functionally.

Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore propagated GAS in HA-enriched growth media in an attempt to create a simple biological system that could reflect some elements of GAS pathogenesis. Our results show that several recognised virulence factors were up-regulated in HA-enriched media, including the M1 protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential GAS virulence factors.

The ability to efficiently produce hundreds of proteins in parallel is the most basic requirement of many aspects of proteomics. Overcoming the technical and financial barriers associated with high throughput protein production is essential for the development of an experimental platform to query and browse the protein content of a cell (e.g. protein and antibody arrays). Proteins are inherently different one from another in their physicochemical properties; therefore, no single protocol can be expected to successfully express most of the proteins. Instead of optimizing a protocol to express a specific protein, we used sequence analysis tools to estimate the probability of a specific protein to be expressed successfully using a given protocol, thereby avoiding a priori proteins with a low success probability. A set of 547 proteins, to be used for antibody production and selection, was expressed in Escherichia coli using a high throughput protein production pipeline. Protein properties derived from sequence alone were correlated to successful expression, and general guidelines are given to increase the efficiency of similar pipelines. A second set of 68 proteins was expressed to investigate the link between successful protein expression and inclusion body formation. More proteins were expressed in inclusion bodies; however, the formation of inclusion bodies was not a requirement for successful expression.

Here, we have set out to demonstrate that PROTEOMICS in its various forms has demonstrated its utility across the entire drug development pipeline from initial TARGET discovery and validation through to monitoring drug responses during clinical trials. In recent times, proteomic techniques are also increasingly making their impact felt in the very last step in this pipeline, namely process validation during the production phase. This more generalized impact has only become possible as a result of improved reproducibility, sensitivity, user-friendliness and throughput of the techniques employed. The next phase of genomics is now generally hailed as focusing on PROTEOMICS. These techniques are evolving rapidly to meet the demands of the scientific and pharmaceutical communities. Such specialized approaches as chemoproteomics are likely to usher in still further advances in global screening of biomolecule binding to therapeutic agents and high-throughput characterization on line via mass spectrometry of these binders. The results of such analyses can then be fed to in silico analysis so as to better understand the shared structural motifs and domains that have given rise to drug binding across a wide spectrum of cellular components. In such a manner, PROTEOMICS data are likely to be seen increasingly to actually drive many aspects of future drug development and improved safety of therapeutic agents, be they traditional small molecules or biomolecules.

Research activities centered on the ensemble of and individual human proteins have taken on numerous guises, some of which fall under the banner of what could be defined as a Human Proteome Project (HPP). However, the latter has yet to take-on the apparent global focus of its predecessor, the Human Genome Project. The reasons for this are both financial and technical. The disparate properties afforded to each protein by a 20-letter code render a single unifying approach difficult to implement, while the current limit of analytical detection has yet to deliver an entire proteome for even the simplest of microbes. The situation is complicated further by the fact that low abundance proteins dominate within any living cell. Thus, enhancement of signal-to-noise ratio by affinity ligands becomes of paramount importance if whole-organism proteomics is to be realized. The generation of such ligands (molecules exhibiting desirable affinity and selectivity for target) could provide the necessary focus and a task list with a definable beginning and end. Such a finite task list is considered essential if an HPP might one day deliver global coverage on a scale seen currently for the total DNA sequence of some 200 living organisms.

Proteome studies complement current molecular approaches through analysis of the actively translated portion of the genome (the "functional proteome"). Two-dimensional gel electrophoresis (2-DGE) utilising immobilized pH gradients of pH 2.3-5.0 and pH 6.0-11.0, developed with predetermined regions of overlap compatible with commercially available pH 4.0-7.0 gradients, permitted the display of a significant portion of the proteome of Mycobacterium tuberculosis H37Rv. A significant portion of the M. tuberculosis proteome, in the molecular mass (M(r)) window 5 kDa to 200 kDa and with isoelectric point (pI) between pH 2.3 and 11.0, was visualised for the first time. A total of 493 protein spots were effectively resolved, including 126 spots that could not be seen using standard pH 4.0-7.0 gradients. These results were used to compare the physical properties of the observed proteins to the theoretical predictions of the recently completed M. tuberculosis H37Rv genome. Most proteins were found in the pI and mass window of pH 4.0-7.0 and 10-100 kDa. Analysis of the predicted proteome revealed a bimodal pI distribution, with substantial numbers of proteins in the pI regions 4.0-7.0 and 9.0-12.0 as has been seen for the majority of completed genomes. Such data may reveal current limitations in experimental extraction and separation of extremely basic, high M(r) and hydrophobic proteins via 2-DGE. Conversely, 13 acidic proteins were observed with pI less than the lowest value predicted by the genome. In addition, a subset of small protein (< 10 kDa) were observed within the pI region of pH 5.0-8.0 that were not predicted by the complete genomic sequence, reflecting the current inability to distinguish small genes from within DNA sequence. This work represents the foundation for comparing the protein expression patterns of different pathogenic and nonpathogenic M. tuberculosis strains. The characterization of M. tuberculosis protein expression, further facilitated by the recent completion of the genome sequence, could aid in developing more effective diagnostic or therapeutic reagents.

Forty-two protein spots of observed M(r) 6-15 kDa were resolved by two-dimensional gel electrophoresis, stained by Coomassie blue and subjected to Edman microsequencing. All of the proteins could be related back to their encoding open reading frames, thereby vindicating the bioinformatic tools currently utilised in their identification. However, only 14/42 gene-products were expressed as annotated. Translation was confirmed for 14 open reading frames with no attributed function (EcoGene Y-entries), while N-terminal sequence allowed the start codon to be accurately annotated for the genes yigF, yccU, yqiC, ynfD, and yeeX. The methionine start codon was cleaved in 11 gene-products (AtpE, Hns, RpoZ, RplL, CspC, YccJ, YggX, YjgF, HimA, InfA, RpsQ) and a further five showed loss of a signal peptide (PspE, HdeB, HdeA, YnfD, YkfE). Internal (Tig, AtpA, TufA) and N-terminal fragmentation (CspD, RpsF, AtcU) of much larger proteins was also detected, which may have resulted from physiological or translational processes. M(r) and pI isoforms were detected respectively for PtsH and GatB, each being phosphoproteins, as well as RplY which manifested differences with respect to predicted M(r) and pI. In addition, YjgF was shown to belong to a small gene family of unknown function with ancient conserved regions across procaryotes and eucaryotes. YgiN was revealed to have a paralogue and orthologues in Bacillus subtilis, Synechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoea, and Rhodococcus erythropolis. Orthologues are also reported for YihD, YccU and YeeX. Of the 14 Y-genes, only YkfE possessed no detectable orthologues. These results highlight the need to complement genomic analysis with detailed proteomics in order to gain a better understanding of cellular molecular biology, while the confirmation of the open reading frame start codon using Edman degradation protein microsequencing has yet to be superseded by recent advances in mass spectrometry.

Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette-Guerin (BCG) vaccine in recent trials has prompted a search for potential replacements. Recent advances in molecular and cell biology have cast doubts on the ability of genetic analysis alone to predict polygenic human diseases and other complex phenotypes and have therefore redirected our attention to proteome studies to complement information obtained from DNA sequencing initiatives. Novel acidic (pH 2.3-5) and basic (pH 6-11) IPG gel gradients were employed in conjunction with commercially available pH 4-7 gradients to significantly increase (fourfold) the number of protein spots previously resolved on two-dimensional (2-D) gels of Mycobacterium species. A total of 772 and 638 protein spots were observed for M. bovis BCG and M. tuberculosis H37Rv, respectively, the latter corresponding to only the pH regions 4-7 and 6-11. Of interest was the bimodal distribution observed for proteins separated from M. bovis BCG across both M(r) and pH ranges. Some differences in protein expression were observed between these two organisms, contrary to what may have been expected considering the high degree of conservation in gene order and sequence similarity between homologous genes. Further work will be directed towards a more detailed analysis of these differences, so as to allow more accurate diagnosis between vaccination and active tuberculosis. The latter is of major importance to epidemiological studies and for patient management.

The most extensive linear pH gradients yet employed in combination with two-dimensional gel electrophoresis are described, along with their application in proteome analysis. A significant proportion of the protein compliment of bacterial species is believed to be accessible using an extended linear pH gradient of 2.3 to 11.0. Protein standards with predicted isoelectric points (pI) ranging from 3.24 to 9.56 were used to confirm focusing positions with respect to the immobilised pH gradients (IPG) prior to mapping studies of Ochrobactrum anthropi. Multiple gel images were used to construct contiguous windows of protein expression ('proteomic contigs') within 18 cm pH gradients 2.3-5, 4-7, and 6-11 in conjunction with 15% T and 7.5% T acrylamide gels, the latter being used to resolve higher molecular weight (M(r)) proteins. Each IPG had a 5 cm region of similar pH gradient overlap at pH 4-5 and pH 6-7 that was used to construct an image of protein expression characteristic of whole cell lysates. This is reminiscent of genomic sequencing initiatives whereby portions are combined to form a contiguous picture of the whole. The protein maps obtained demonstrated a means of resolving the many tens of thousands of cellular proteins likely to occur in eukaryotic systems, but also highlighted the need to further optimise protein extraction, equilibration buffers, and separation conditions of higher M(r) proteins occurring at extreme pI. Theoretical 2-D protein maps were constructed for five organisms for which the total DNA sequence is now available. In all cases, higher M(r) acidic and basic proteins were shown to be common.

A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.

The ability to effectively search databases for the identification of protein spots from two-dimensional electrophoresis gels has become an essential step in the study of microbial proteomes. A variety of analytical techniques are currently being employed during protein characterisation. A number of algorithms used to search databases, accessible via the World Wide Web, depend upon information concerning N- and C-terminal microsequence, amino acid composition, and peptide-mass fingerprinting. The effectiveness of nine such algorithms, as well as COMBINED (software developed in this laboratory for identifying proteins across species boundaries) was examined. Fifty-four ribosomal proteins from the Mycoplasma genitalium genome, and 72 amino acyl tRNA synthetases from the Haemophilus influenzae, M. genitalium and Methanococcus jannaschii genomes were chosen for study. These proteins were selected because they represent a wide range of sequence identities across species boundaries (22.7-100% identity), as detected by standard sequence alignment tools. Such sequence variation allowed for a statistical comparison of algorithm success measured against published sequence identity. The ability of analytical techniques used in protein characterisation and associated database query programs to detect identity at the functional group level was examined for proteins with low levels of homology at the gene/protein sequence level. The significance of these theoretical data manipulations provided the means to predict the utility of data acquired experimentally for non-sequence-dependent software in proteome analysis. The data obtained also predicted that 'sequence tagging' of peptide fingerprints would need to be accompanied by at least 11-20 residues of amino acid sequence for it to be widely used for protein characterisation across species boundaries.

In muscle cells actin exists as a mixture of monomeric (G-actin) and filamentous actin (F-actin) and ionic conditions strongly favor the formation of F-actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G-actin, the so-called G-actin-binding proteins (G-ABPs). We have coupled monomeric actin to divinylsulphone-activated agarose (Mini-Leak) to isolate G-ABPs in human skeletal muscle. Eluted proteins were analyzed by two-dimensional gel electrophoresis (2-DE), which shows that some proteins are selectively retained. Deoxyribonuclease I (DNase I) is known to bind residues at the "pointed end" of actin (subdomains 2 and 4) with a high affinity. When DNase I is bound to the actin Mini-Leak before applying the skeletal muscle extract, the 2-DE gels of the eluted proteins reveals differences when compared to gels of proteins eluted from actin-Mini-Leak and DNase I-Mini-Leak affinity columns. This strategy should detect ABPs which bind to sites other than the DNase I-binding site and some may prove to be novel.

(1996). Progress with Proteome Projects: Why all Proteins Expressed by a Genome Should be Identified and How To Do It. Biotechnology and Genetic Engineering Reviews: Vol. 13, No. 1, pp. 19-50.

A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of ‘hierarchical’ analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or ‘proteome’ is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.

A set of 1438 human exons was subjected to nested PCR. The initial success rate using a standard PCR protocol required for ligation-independent cloning was 83.4%. Logistic regression analysis was conducted on 27 primer- and template-related characteristics, of which most could be ignored apart from those related to the GC content of the template. Overall GC content of the template was a good predictor for PCR success; however, specificity and sensitivity values for predicted outcome were improved to 84.3 and 94.8%, respectively, when regionalized GC content was employed. This represented a significant improvement in predictability with respect to GC content alone (P < 0.001; chi(2)) and is expected to increase in relative sensitivity as template size increases. Regionalized GC was calculated with respect to a threshold of 61% GC content and a sliding window of 21 bp across the target sequence. Fine-tuning of PCR conditions is not practicable for all target sequences whenever a large number of genes of different lengths and GC content are to be amplified in parallel, particularly if total open reading frame or domain coverage is essential for recombinant protein synthesis. Thus, the present method is proposed as a means of grouping subsets of genes possessing potentially difficult target sequences so that PCR conditions can be optimized separately in order to obtain improved outcomes.

Herpetostrongylinae is redefined. A Woolleya-like parasite in Gondwanaland didelphoids is proposed as the ancestral stock of the Herpetostrongylinae parasitic in Australian marsupials. Three evolutionary lines are postulated for the radiation within the Herpetostrongylinae, each derived from a form resembling existing species of Woolleya Mawson, 1973, parasitic in Dasyuridae and Thylacinidae: (i) a short lineage restricted to Dasyuridae; (ii) Dasyuridae-Peramelidae and Myrmecobiidae, followed by re-invasion into Dasyuridae; (iii) Dasyuridae-Macropodidae-Potoroidae, Petauridae and Phalangeridea. W. hydromyos Mawson, 1973, and Paraustrostrongylus ratti Obendorf, 1979, are interpreted as secondary transfers of marsupial parasites to recent murid invaders. No evidence was found to support peramelid parasites giving rise to species parasitic in Diprotodontia. Herpetostrongylus and Vaucherus are considered as a distinct phylogenetic unit based on south-east Asia and parasitic in reptiles.