ISSN:0173-0835    

DOI:10.1002/elps.1150180802

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe introduction of methods for automated DNA sequence analysis nearly a decade ago, together with more recent advances in the field of bioinformatics, have revolutionized biology and medicine and have ushered in a new era of genomic science, the study of genes and genomes. These new technologies have had an impact on many areas of research, including the association between genes and disease, in DNA‐based diagnostics, and in the sequencing of genomes from human and other model organisms. The demonstration in 1995, that automated DNA sequencing methods could be used to decipher the entire genome sequence of a free‐living organism,Haemophilus influenzae, was a milestone in both the genomics and microbial fields [1]. Since the first report of the complete sequence ofH. influenzae, these methodologies have been adopted by laboratories around the world. The complete genomic sequence of five eubacterial species [1–5], one archaea [6], and the eukaryote,Saccharomyces cerevisiae[7], have been reported in the last 18 months. At the beginning of 1997 more than a dozen microbial genome projects are at or near completion, with many others in progress. It is likely that in the next few years we will see the complete sequence of perhaps as many as 30–40 microbial genomes. In this article, we will review methods for whole genome sequencing and analysis and examine how this information can be exploited to better understand microbial physiology and ev

ISSN:0173-0835    

DOI:10.1002/elps.1150180803

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA 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 proble

ISSN:0173-0835    

DOI:10.1002/elps.1150180804

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe gene‐protein database ofEscherichia coliis a collection of data, largely generated from the separation of complex mixtures of cellular proteins on two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The database currently contains about 1600 protein spots. The data are comprised of both identification information for many of these proteins and data on how the level or synthesis rates of proteins vary under different growth conditions. Three projects are underway to further elucidate theE. coliproteome including a project to localize on 2‐D gels all of the open reading framed encoded by theE. colichromosome, a project to determine the condition(s) under which each open reading frame is expressed and a project to determine the abundance and location of each protein in the cell. Applications for proteome databases for cell modeling are discussed and examples of applications in therapeutic drug discovery ar

ISSN:0173-0835    

DOI:10.1002/elps.1150180805

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractFollowing the complete sequencing of the genome of the univellular cyanobacterium,Synechocystissp. strain PCC6803, within our institute, a protein‐gene linkage map of this photosynthetic microorganism was successfully constructed for 130 high abundance proteins present on two‐dimensional gels. An additional six proteins were analyzed, but were probably encoded extrachromosomally. In order to demonstrate the usefulness of this protein‐gene linkage map, we analyzed the changes that occur in cellular proteins after illumination of PCC6803 cells. The results indicate that this protein‐gene linkage map greatly simplifies the identification process of such modulated genes. After illumination, at least three distinctive spots with reduced intensity were detected on two‐dimensional gels and the corresponding genes of two of these were successfully identified as chaperonin 2 and aTortula ruralisrehydrin‐related gene. Thus, the combination of the protein‐gene linkage map and two‐dimensional gel electrophoresis should permit a comprehensive analyses of the proteins encoded by the genome (i.e., “proteome”) of this photosynthetic autotroph. This post‐genome project represents a productive way of exploiting the information obtained from the sequencing of the

ISSN:0173-0835    

DOI:10.1002/elps.1150180806

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractMining the emerging abundance of microbial genome sequences for hypotheses is an exciting prospect of “functional genomics”. At the forefront of this effort, we compared the predictions of the completeEscherichia coligenomic sequence with the observed gene products by assessing 381 proteins for their matureN‐termini,in vivoabundances, isoelectric points, molecular masses, and cellular locations. Two‐dimensional gel electrophoresis (2‐DE) and Edman sequencing were combined to sequence Coomassie‐stained 2‐DE spots representing the abundant proteins of wild‐typeE. coliK‐12 strains. Greater than 90% of the abundant proteins in theE. coliproteome lie in a small isoelectric point and molecular mass window of 4–7 and 10–100 kDa, respectively. We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone. Of the 223 uniquely identified loci, 60% of the encoded proteins are proteolytically processed. As previously reported, the initiator methionine was efficiently cleaved when the penultimate amino acid was serine or alanine. In contrast, when the penultimate amino acid was threonine, glycine, or proline, cleavage was variable, and valine did not signal cleavage. Although signal peptide cleavage sites tended to follow predicted rules, the length of the putative signal sequence was occassionally greater than the consensus. For proteins predicted to be in the cytoplasm or inner membrane, theN‐terminal amino acids were highly constrained compared to proteins localized to the periplasm or outer membrane. Although cytoplasmic proteins follow theN‐end rule for protein stability, proteins in the periplasm or outer membrane do not follow this rule; several haveN‐terminal amino acids predicted to destabilize the proteins. Surprisingly, 18% of the identified 2‐DE spots represent isoforms in which protein products of the same gene have different observed pIandMr, suggesting they are post‐translationally processed. Although most of the predicted and observed values for isoelectric point and molecular mass show reasonable concordance, for several proteins the observed values significantly deviate from the expected values. Such discrepancies may represent either highly processed proteins or misinterpretations of the genomic sequence. Our data suggest that AhpC, CspC, and HdeA exist as covalent homomultimers, and that IcdA exists as at least three isoforms even under conditions in which covalent modification is not predicted. We enriched for proteins based on subcellular location and found several proteins in u

ISSN:0173-0835    

DOI:10.1002/elps.1150180807

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractWith the completion of theHaemophilus influenzaeRd genomic sequence, we know the identity of most of the theoretical proteins in the proteome of this bacterium. However, the most abundant components of the actual proteome are unknown. Using mass spectrometry and two‐dimensional gel electrophoresis (2‐DE), we sequenced and analyzed the most abundant proteins observed in the ATCC reference strain ofH. influenzae, NCTC 8143 (303 of ≈︁ 400 Coomassie‐stained 2‐DE spots). To automate the identification of 2‐DE spots, we coupled a liquid autosampler to a microcolumn liquid chromatography electrospray ionization tandem mass spectrometer capable of identifying 22 spots per day. From the 303 sequenced spots, we identified 263 unique proteins. Most of the abundant proteins lie in an isoelectric point range of pH 4–7 and a molecular mass range of 10–100 kDa. Of the observed proteins, the most abundant is the outer membrane protein P2. Based on variety and abundance, proteins involved in energy metabolism and macromolecular synthesis are the dominant classes of proteins. Unexpectedly, tryptophanase was identified as a highly abundant protein in the strain NCTC 8143 whose sequence is rot present in the genome of the Rd strain. By searching the tandem mass spectra against the translated genomic sequence, we identified several proteins which were not annotated in the genomic sequence. Surprisingly, 22% of the identified 2‐DE spots represent isoforms in which gene products with the same primary sequence have different observed pIandMr, indicating that these proteins are post‐translationally processed. Although most proteins' predicted and observed isoelectric points and molecular masses show reasonable concordance, the observed values for several proteins deviate significantly from the predicted values. These anomalies may represent either highly processed proteins or misinterpretations of the genomic sequence. Using the technology developed in this project, the protein expression of other strains ofH. influenzaegrown under different environmental conditions can be compared to identify differenc

ISSN:0173-0835    

DOI:10.1002/elps.1150180808

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractSpiroplasma 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 ofS. melliferumwas 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) fromS. melliferumwere further characterised by one, or a combination, of the following: amino acid analysis, peptide‐mass fingerprintingviamatrix assisted laser desorption ionisation‐time of light (MALDI‐TOF) mass spectrometry, andN‐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. Geneproducts from major families such as glycolysis, translation, transcription, cellular processes, energy metabolism and protein synthesis were identified. Several gene‐products characterised inS. melliferumwere not previously found in studies of the entireMycoplasma genitaliumandMycoplasma pneumoniae(both closely related Mollicutes) genomes. The presence of such geneproducts inS. melliferumis discussed in terms of genome size as compared with the smallest known free‐living organisms. Finally, the levels of expression ofS. melliferumgene‐products were determined with respect to total optical itensity associated with all visible proteins expre

ISSN:0173-0835    

DOI:10.1002/elps.1150180809

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractTwo‐dimensional (2‐D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2‐D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence‐derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2‐D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2‐D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2‐D gel patterns. Using the 2‐D gel map and the information available in YDP, 2‐D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature‐sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post‐translational modification (N‐terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more protein

ISSN:0173-0835    

DOI:10.1002/elps.1150180810

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractProteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function. This is achieved through the combination of two technologies: protein separation and selection by two‐dimensional gel electrophoresis, and protein identification and characterization by mass spectrometry. This methodological outline sketches the strengths and weaknesses of the two central technologies used, and provides both practical tips and the theoretical background for their utilization. One application of these technologies is illustrated by the characterization of genes, revealed by sequencing, but which have no — or only weak homology — to any other known genes. Other applications, for example the identification of protein markers for particular human diseases, are only referred to. The aim of the article is thus to provide the basis for a sound understanding of the full potential and limitations of proteome ana

ISSN:0173-0835    

DOI:10.1002/elps.1150180811

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY