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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.

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.

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.

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.

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.