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I Humphery-Smith

Publications and source records attributed to I Humphery-Smith.

At least 19 recordsLinked to original sources
PubMed2000

The proteome of Mycoplasma genitalium. Chaps-soluble component.

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.

Bacterial Proteins↗
PubMed1999

Cross-species characterisation of abundantly expressed Ochrobactrum anthropi gene products.

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.

Acetyl-CoA C-Acetyltransferase↗
PubMed1998

Small genes/gene-products in Escherichia coli K-12.

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.

Amino Acid Sequence↗
PubMed1998

Comparison of predicted and observed properties of proteins encoded in the genome of Mycobacterium tuberculosis H37Rv.

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.

Bacterial Proteins↗
PubMed1998

Low molecular weight proteins: a challenge for post-genomic research.

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.

Amino Acid Sequence↗
PubMed1998

[Hemoglobin alpha chain isoelectric point modification under the action of urea, sodium cyanate, succinic anhydride or diethylene triamine pentaacetic acid anhydride].

A monomeric protein, the hemoglobin alpha chain, was used to compare four protocols for conjugation with diethylene triamine pentaacetic (DTPA) anhydride. Carbamylation and succinylation were also performed. The isoelectric point (pI) was 7.7 for the native protein versus only 5.5 to 7.3 for the five carbamylated derivatives and 4.0 to 7.0 for the six succinylated derivatives. With carbamylation or succinylation, increasing the molar ratio (agent/protein) was associated with a gradual downward pI shift producing trains of bands. This phenomenon did not occur with DTPA conjugation, whose results varied with the method used; only one derivate (pI 6.7) was produced by all four methods, and multiple fine bands with pH values in the vicinity of 3.6 were seen. For the protein, the pI shift varied with the number of groups inserted on the primary amine residues. Also, the shift was larger if the inserted groups carried electrically-charged moieties.

Chelating Agents↗
PubMed1997

Malate/lactate dehydrogenase in mollicutes: evidence for a multienzyme protein.

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.

Amino Acid Sequence↗
PubMed1997

Conserved motifs as the basis for recognition of homologous proteins across species boundaries using peptide-mass fingerprinting.

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.

Amino Acid Sequence↗
PubMed1997

Two-dimensional gel electrophoresis of actin-binding proteins isolated by affinity chromatography from human skeletal muscle.

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.

Absorption↗
PubMed1997

Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.

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.

Animals↗
PubMed1997

Proteome analysis of Spiroplasma melliferum (A56) and protein characterisation across species boundaries.

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 Sequence↗
PubMed1997

Proteomic 'contigs' of Ochrobactrum anthropi, application of extensive pH gradients.

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.

Bacterial Proteins↗
PubMed1997

'Proteomic contigs' of Mycobacterium tuberculosis and Mycobacterium bovis (BCG) using novel immobilised pH gradients.

Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette-Guérin (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.

Bacterial Proteins↗
PubMed1997

Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients.

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.

Amino Acid Sequence↗
PubMed1997

Peptide-mass fingerprinting and the ideal covering set for protein characterisation.

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.

Amino Acid Sequence↗
PubMed1997

Evaluation of algorithms used for cross-species proteome characterisation.

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.

Algorithms↗