Phil Cash - Research

Phil Cash
Dr. Phil Cash
Department of Medical Microbiology
University of Aberdeen

Telephone: 01224-555809
email: p.cash@abdn.ac.uk

Principal Research Interests

Selected Publications

Related Research links at Aberdeen

Principal Research Interests

My principal area of research is on the use of proteomics in the biomedical sciences, specifically the analysis of human bacterial pathogens. The proteome describes the total protein complement expressed by a cell at any one time. It is a highly dynamic entity that can change in response to the intracellular and extracellular environment. Since analyses carried out at the level of the cellular proteins can provide information on post-translational events, data not readily derived from the genome sequence alone, proteomics provides an ideal complementary approach to traditional genomic based methods for investigating gene expression and cellular metabolism. The relative simplicity of bacteria, combined with the extensive genomic data that are now available, makes the study of bacterial proteomes a fruitful area of research. It is reasonable to expect that the combined data from proteomics and genomics will provide us with a comprehensive picture of bacterial cell metabolism in the near future. Many different methods can be used to investigate cellular proteomes and my laboratory has concentrated on the use of 2-Dimensional Electrophoresis (2DE) for protein resolution and peptide mass mapping for subsequent identification of the resolved proteins, the latter using the facilities of the Aberdeen Proteome Facility. 2DE separates proteins on the basis of their native charge (first dimension) and molecular mass (second dimension) and can provide both qualitative and quantitative data on protein synthesis. In peptide mass mapping the resolved proteins are digested with trypsin and the derived peptides accurately sized using mass spectrometry. These peptide masses can then be used to search the peptide profiles predicted for sequenced proteins to identify the experimentally identified proteins. Using these two methods proteome databases can be produced. Figure 1 shows some of our data for the Haemophilus influenzae proteome. Similar studies are in progress for Streptococcus pneumoniae and Helicobacter pylori.

Figure 1

Proteome Maps developed for Haemophilus influenzae by 2-Dimensional Electrophoresis

Click here for full sized image

Proteomics plays a key role as one of the tools in Functional Genomics. Figure 2 illustrates the general approach for the functional analysis of bacterial proteomes. Essentially, a bacterial culture is exposed to an external stimulus (e.g. growth conditions) whilst a second culture is maintained untreated as the control. The bacterial proteins are extracted and then analysed by 2DE. Using dedicated software the amounts of the individual proteins are determined and compared between the two culture conditions. Proteins showing significant differences in their biosynthetic levels are selected and identified by peptide mass mapping. These proteins may then be targeted for further analysis using complementary genomic technologies. Although Figure 2 illustrates the analysis of the bacterial response to an external environmental stimulus, the same approach may be used to compare pathogenic and non-pathogenic bacteria.

Figure 2

Functional Analysis of Bacterial Proteomes

Click here for full sized image

In collaborative studies with Dr. Hamish McKenzie (Department of Medical Microbiology) we have used proteomics to examine the basis of erythromycin resistance in S. pneumoniae. The approach used is shown in Figure 3. Sensitive and resistant clinical isolates of S. pneumoniae were compared by 2DE to locate characteristic protein differences associated with antibiotic resistance. A characteristic change in the expression of a bacterial protein was located and identified as glyceraldehdye-3-phosphate dehydrogenase (GAPDH) by homology with other Streptococci. Dr. Phil Carter (Department of Medical Microbiology) designed oligonucleotide primers to sequence this gene in the original collection of S. pneumoniae isolates and demonstrated the occurence of a specific nucleotide substitution that correlated with both antibiotic resistance and the proteomic change. The significance of the GAPDH variation in antibiotic resistance and bacterial physiology is under investigation.

Figure 3

Analysis of Antibiotic Resistance in Streptococcus pneumonia

Click here for full sized image

Although a significant proportion of our research concerns bacterial proteomes, we are applying similar approaches to other problems in biomedical research. Much of this latter work is through collaborative work developed as part of the Aberdeen Proteome Facility.

Top

Selected Publications

Gow, J., Cash, P., Behan, W. H., McGarry, F., Simpson, K., and Behan, P. O.(1995). Detection of picornavirus genomic and template RNA strands by a novel semi-nested polymerase chain reaction-technique and agarose gel electrophoresis. Electrophoresis 16, 338-340. [Medline]

Abadi, F. J., Carter, P. E., Cash, P., and Pennington, T. H.(1996). Rifampin resistance in Neisseria meningitidis due to alterations in membrane permeability. Antimicrobial Agents & Chemotherapy 40, 646-651. [Medline]

Gimenez, H. B., Chisholm, S., Dornan, J., and Cash, P.(1996). The neutralising and enhancing activities of human respiratory syncytial virus-specific antibodies. Clin. Diag. Lab. Immunol. 3, 280-286. [Medline]

Gow, J. W., Behan, W. M., Cash, P., Simpson, K., and Behan, P. O. (1997). Genomic and template RNA transcription in a model of persistent enteroviral infection. Journal of Neurovirology 3, 76-82. [Medline]

Cash, P., Argo, E., Langford, P., and Krol, S.J. (1997) Development of an Haemophilus 2D protein database. Electrophoresis 18, 1472-1482 [Medline]

Cash P. (1997) Analysis of Bacterial Protein Synthesis using 2-Dimensional Electrophoresis. in: Perspectives on Protein Engineering 2. (Geisow, M.J., ed.) BIODIGM, Nottingham, UK

Cash, P. (1998) Characterisation of bacterial proteomes by two-dimensional electrophoresis. Analytica Chimica Acta. 372, 121-145

Gimenez, H.B., Cash, P., Laing, R.B.S. and Douglas, J.G. (1999) Cytokine expression and morphology of in vitro grown monocytes from patients with chronic fatigue syndrome. Journal of Chronic Fatigue Syndrome 5, 47-60.

Cash, P., Argo, E., Ford, L., Lawrie, L. and McKenzie, H. (1999) A proteomic analysis of erythromcyin resistance in Streptococcus pneumoniae. Electrophoresis 20, 2259-2268 [Medline]

Cash, P. (2000) Proteomics in Medical Microbiology. Electrophoresis 21, 1187-1201

Chambers G. Lawrie L. Cash P. Murray G.I. (2000) Proteomics: a new approach to the study of disease. Journal of Pathology. 192(3):280-288

Martin, S.A.M., Cash, P., Blaney, S. and Houlihan, D.F. (2001) Proteome analysis of rainbow trout (Oncorhyncus mykiss) liver proteins during short term starvation. Fish Physiology and Biochemistry 24, 259-270

Cash, P. (2002) Proteomics: the protein revolution. Biologist, 49, 58-62

Fowler P.A., Mason H.D., Melvin W.T., Wilson Y., Cash P., Sorsa T., and Harris W.J. (2002) A 60-66 kDa protein with gonadotrophin surge-attenuating factor (GnSAF) bioactivity is produced by human ovarian granulosa cells. Mol. Human Reprod. 8, 823-832

Amezaga M.R., Carter P.E., Cash P., and McKenzie H. (2002) Molecular epidemiology of eryhtromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites. Journal of Clinical Microbiology, 40, 3313-3318

Booth I.R., Cash P. and O'Byrne C.P. (2002) Sensing and adapting to acid stress. Antonie van Leeuwenhoek 81: 33–42,

Cash P. and Kroll J.S. (2003) Protein characterization by two-dimensional gel electrophoresis. Methods in Molecular Medicine. 71:101-118.

Cash, P. (2003) Proteomics of bacterial pathogens. Advances in Biochemical Engineering/Biotechnology (In Press).

Birch R.M., O'Byrne, C, Booth I.R. and Cash P. (2003) Enrichment of Escherichia coli Proteins by Column Chromatography on Reactive Dye Columns. Proteomics (In Press)

Top

Related Research Links at Aberdeen

Microbial Proteomes - Laboratory website presenting further information on our work and details of the methods we use.

Aberdeen Proteome Facility - The provision of a classical proteomics service (2D electrophoresis, peptide mass mapping and Edman sequencing) for academic and commercial users.

Top

Last Updated: 30th January 2003

	Dr. Phil Cash Department of Medical Microbiology University of Aberdeen Telephone: 01224-555809 email: p.cash@abdn.ac.uk
	Principal Research Interests Selected Publications Related Research links at Aberdeen
	Principal Research Interests My principal area of research is on the use of proteomics in the biomedical sciences, specifically the analysis of human bacterial pathogens. The proteome describes the total protein complement expressed by a cell at any one time. It is a highly dynamic entity that can change in response to the intracellular and extracellular environment. Since analyses carried out at the level of the cellular proteins can provide information on post-translational events, data not readily derived from the genome sequence alone, proteomics provides an ideal complementary approach to traditional genomic based methods for investigating gene expression and cellular metabolism. The relative simplicity of bacteria, combined with the extensive genomic data that are now available, makes the study of bacterial proteomes a fruitful area of research. It is reasonable to expect that the combined data from proteomics and genomics will provide us with a comprehensive picture of bacterial cell metabolism in the near future. Many different methods can be used to investigate cellular proteomes and my laboratory has concentrated on the use of 2-Dimensional Electrophoresis (2DE) for protein resolution and peptide mass mapping for subsequent identification of the resolved proteins, the latter using the facilities of the Aberdeen Proteome Facility. 2DE separates proteins on the basis of their native charge (first dimension) and molecular mass (second dimension) and can provide both qualitative and quantitative data on protein synthesis. In peptide mass mapping the resolved proteins are digested with trypsin and the derived peptides accurately sized using mass spectrometry. These peptide masses can then be used to search the peptide profiles predicted for sequenced proteins to identify the experimentally identified proteins. Using these two methods proteome databases can be produced. Figure 1 shows some of our data for the Haemophilus influenzae proteome. Similar studies are in progress for Streptococcus pneumoniae and Helicobacter pylori.
		Figure 1 Proteome Maps developed for Haemophilus influenzae by 2-Dimensional Electrophoresis Click here for full sized image
	Proteomics plays a key role as one of the tools in Functional Genomics. Figure 2 illustrates the general approach for the functional analysis of bacterial proteomes. Essentially, a bacterial culture is exposed to an external stimulus (e.g. growth conditions) whilst a second culture is maintained untreated as the control. The bacterial proteins are extracted and then analysed by 2DE. Using dedicated software the amounts of the individual proteins are determined and compared between the two culture conditions. Proteins showing significant differences in their biosynthetic levels are selected and identified by peptide mass mapping. These proteins may then be targeted for further analysis using complementary genomic technologies. Although Figure 2 illustrates the analysis of the bacterial response to an external environmental stimulus, the same approach may be used to compare pathogenic and non-pathogenic bacteria.
		Figure 2 Functional Analysis of Bacterial Proteomes Click here for full sized image
	In collaborative studies with Dr. Hamish McKenzie (Department of Medical Microbiology) we have used proteomics to examine the basis of erythromycin resistance in S. pneumoniae. The approach used is shown in Figure 3. Sensitive and resistant clinical isolates of S. pneumoniae were compared by 2DE to locate characteristic protein differences associated with antibiotic resistance. A characteristic change in the expression of a bacterial protein was located and identified as glyceraldehdye-3-phosphate dehydrogenase (GAPDH) by homology with other Streptococci. Dr. Phil Carter (Department of Medical Microbiology) designed oligonucleotide primers to sequence this gene in the original collection of S. pneumoniae isolates and demonstrated the occurence of a specific nucleotide substitution that correlated with both antibiotic resistance and the proteomic change. The significance of the GAPDH variation in antibiotic resistance and bacterial physiology is under investigation.
		Figure 3 Analysis of Antibiotic Resistance in Streptococcus pneumonia Click here for full sized image
	Although a significant proportion of our research concerns bacterial proteomes, we are applying similar approaches to other problems in biomedical research. Much of this latter work is through collaborative work developed as part of the Aberdeen Proteome Facility. Top
	Selected Publications Gow, J., Cash, P., Behan, W. H., McGarry, F., Simpson, K., and Behan, P. O.(1995). Detection of picornavirus genomic and template RNA strands by a novel semi-nested polymerase chain reaction-technique and agarose gel electrophoresis. Electrophoresis 16, 338-340. [Medline] Abadi, F. J., Carter, P. E., Cash, P., and Pennington, T. H.(1996). Rifampin resistance in Neisseria meningitidis due to alterations in membrane permeability. Antimicrobial Agents & Chemotherapy 40, 646-651. [Medline] Gimenez, H. B., Chisholm, S., Dornan, J., and Cash, P.(1996). The neutralising and enhancing activities of human respiratory syncytial virus-specific antibodies. Clin. Diag. Lab. Immunol. 3, 280-286. [Medline] Gow, J. W., Behan, W. M., Cash, P., Simpson, K., and Behan, P. O. (1997). Genomic and template RNA transcription in a model of persistent enteroviral infection. Journal of Neurovirology 3, 76-82. [Medline] Cash, P., Argo, E., Langford, P., and Krol, S.J. (1997) Development of an Haemophilus 2D protein database. Electrophoresis 18, 1472-1482 [Medline] Cash P. (1997) Analysis of Bacterial Protein Synthesis using 2-Dimensional Electrophoresis. in: Perspectives on Protein Engineering 2. (Geisow, M.J., ed.) BIODIGM, Nottingham, UK Cash, P. (1998) Characterisation of bacterial proteomes by two-dimensional electrophoresis. Analytica Chimica Acta. 372, 121-145 Gimenez, H.B., Cash, P., Laing, R.B.S. and Douglas, J.G. (1999) Cytokine expression and morphology of in vitro grown monocytes from patients with chronic fatigue syndrome. Journal of Chronic Fatigue Syndrome 5, 47-60. Cash, P., Argo, E., Ford, L., Lawrie, L. and McKenzie, H. (1999) A proteomic analysis of erythromcyin resistance in Streptococcus pneumoniae. Electrophoresis 20, 2259-2268 [Medline] Cash, P. (2000) Proteomics in Medical Microbiology. Electrophoresis 21, 1187-1201 Chambers G. Lawrie L. Cash P. Murray G.I. (2000) Proteomics: a new approach to the study of disease. Journal of Pathology. 192(3):280-288 Martin, S.A.M., Cash, P., Blaney, S. and Houlihan, D.F. (2001) Proteome analysis of rainbow trout (Oncorhyncus mykiss) liver proteins during short term starvation. Fish Physiology and Biochemistry 24, 259-270 Cash, P. (2002) Proteomics: the protein revolution. Biologist, 49, 58-62 Fowler P.A., Mason H.D., Melvin W.T., Wilson Y., Cash P., Sorsa T., and Harris W.J. (2002) A 60-66 kDa protein with gonadotrophin surge-attenuating factor (GnSAF) bioactivity is produced by human ovarian granulosa cells. Mol. Human Reprod. 8, 823-832 Amezaga M.R., Carter P.E., Cash P., and McKenzie H. (2002) Molecular epidemiology of eryhtromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites. Journal of Clinical Microbiology, 40, 3313-3318 Booth I.R., Cash P. and O'Byrne C.P. (2002) Sensing and adapting to acid stress. Antonie van Leeuwenhoek 81: 33–42, Cash P. and Kroll J.S. (2003) Protein characterization by two-dimensional gel electrophoresis. Methods in Molecular Medicine. 71:101-118. Cash, P. (2003) Proteomics of bacterial pathogens. Advances in Biochemical Engineering/Biotechnology (In Press). Birch R.M., O'Byrne, C, Booth I.R. and Cash P. (2003) Enrichment of Escherichia coli Proteins by Column Chromatography on Reactive Dye Columns. Proteomics (In Press) Top
	Related Research Links at Aberdeen Microbial Proteomes - Laboratory website presenting further information on our work and details of the methods we use. Aberdeen Proteome Facility - The provision of a classical proteomics service (2D electrophoresis, peptide mass mapping and Edman sequencing) for academic and commercial users. Top
Last Updated: 30th January 2003