Characterisation of <i>Haemophilus influenzae</i> proteins by two‐dimensional gel electrophoresis

Cash, Phillip; Argo, Evelyn; Bruce, K D

doi:10.1002/elps.1150160123

Cited by 47 publications

(43 citation statements)

References 20 publications

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“…The calibration gel strip was stained in parallel with the gels being analysed using colloidal Coomassie Brilliant Blue G-250. The isoelectric points and apparent molecular masses of the proteins were determined by coelectrophoresis with standard protein markers [21].…”

Section: Analysis Of Bacterial Proteins By 2-d Pagementioning

confidence: 99%

“…Unless stated otherwise, soluble proteins were prepared from bacterial cell lysates as described previously [21]. Briefly, single colonies of bacteria were inoculated onto chocolate agar plates and incubated overnight at 37 "C in 5% CO, by which time a confluent bacterial lawn had formed.…”

Section: Preparation Of Soluble Bacterial Proteinsmentioning

confidence: 99%

“…The nontypable reference strain of H. influenzae (designated HI-64443) used as the basis for the 2-D protein database has been described previously [21]. [24].…”

Section: Source Of Bacterial Isolatesmentioning

confidence: 99%

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Development of a Haemophilus two‐dimensional protein database

et al. 1997

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Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135-148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2-D protein profile of the reference nontypable strain of H. influenzae (HI-64443) used as the basis for the 2-D protein database. A composite reference 2-D protein profile of HI-64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI-64443 proteins from the reference 2-D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI-64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2-D protein profile of the Rd strain of H. influenzae was compared to that of HI-64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI-64443 when analysed in parallel. The capacity of 2-D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A "knock-out" mutant in the sodA gene which encodes [Mn]-SOD was characterised with respect to protein synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2-D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.

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Section: Analysis Of Bacterial Proteins By 2-d Pagementioning

confidence: 99%

Section: Preparation Of Soluble Bacterial Proteinsmentioning

confidence: 99%

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Development of a Haemophilus two‐dimensional protein database

et al. 1997

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“…It is also one of the best methods of investigating basic biological processes such as pathogenesis, physiology, and metabolic mechanisms (13,14). In recent years 2-D electrophoresis has been used in proteomic studies of many bacteria, including Haemophilus influenzae (15)(16)(17)(18), Mycobacterium tuberculosis (19 -21), Escherichia coli (13,(22)(23)(24), Helicobacter pylori (25,26), Bacillus subtilis (27,28), Mycoplasma pneumoniae (29), Lactococcus lactis (30 -33), Shigella flexneri (34), Agrobacterium tumefaciens (35), Vibrio cholerae (36), and Brucella melitensis (37).…”

mentioning

confidence: 99%

A Proteome Reference Map and Proteomic Analysis of Bifidobacterium longum NCC2705

Yuan

Zhu²,

Liu³

et al. 2006

Molecular & Cellular Proteomics

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“…[110] tein data has allowed for specific cell fractionation studies to be performed -for example, the identification of six proteins from the spliceosomal U1 small nuclear ribonucleoprotein complex [112] While examination of the E. coli proteome will he greatly assisted by the imminent publication of the total genome sequence, the study of proteomes from closely related bacteria will also benefit. Recent reports have discussed analysis of the total Salmonella typhimurium proteome [ [142]. Separation of proteins on 2-D gels from several Haemophilus species has presented the opportunity to detect those molecules specific to particular phenotypes, for example, pathogenicity 11431.…”

Section: Overview Of Technologymentioning

confidence: 99%

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

Humphery‐Smith

Cordwell

Blackstock³

1997

Electrophoresis

216

111

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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.

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Characterisation of Haemophilus influenzae proteins by two‐dimensional gel electrophoresis

Cited by 47 publications

References 20 publications

Development of a Haemophilus two‐dimensional protein database

Development of a Haemophilus two‐dimensional protein database

A Proteome Reference Map and Proteomic Analysis of Bifidobacterium longum NCC2705

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

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