1. Systems Biology Department of Chemical and Process Engineering The University of Sheffield Phillip Wright

2. Outline Systems biology Proteomics Case study Protein/Peptide separation Mass spectrometry in proteomics and protein/peptide quantification Metabolic reconstruction and networks Other projects Case study: Sulfolobus solfataricus

3. Beecher (2002) Systems Biology Studying physiology of cells on a system wide scale look at the connections between components in cells (quantitative) experimental tools available to begin to look at biological systems

4. How to determine the Relationship between the Genotype and Phenotype ? Reproduced from: K.H. Lee "Proteomics: A Technology-Driven and a Technology-Limited Discovery Science", Trends in Biotechnology 19: 217-222 (2001). In vitro culture design and modelling Kinetics models – metabolic models Population models In vitro system models in vitro modelling and prediction

5. The Cell The Proteome

6. Protein generation Flow diagram of a successful proteomics experiment Protein identification Protein separation Protein quantification Cell growth and protein extraction Electrophoresis and liquid chromatography Mass spectrometry Gel staining and stable isotope labelling Proteomics Work Flow

7. Cell growth and protein extraction Protein generation Genome: 2977 Proteins, 30 % unknown function Cell growth at 80 o C and pH=4 in 50 ml culture Extract proteins by freeze thawing with liquid nitrogen or sonification Result: mixture of hundreds to thousands of proteins Too complex for further processing by mass spectrometry Case study: Proteomic analysis of Sulfolobus solfataricus

8. Gel-based Separation Molecular weight Isoelectric point LC-Based Separation Charge Hydrophobicity Protein/peptide separation Protein separation PI MW The FUTURE – Microfluidic separations

9. pH=3pH=10 2D-Gel Electrophoresis Complex sample 300 spots Excised from the gel Digested with trypsin Analysed with LC-MS- MS 342 proteins identified 2 months Protein separation Protein identification

10. 2D-liquid chromatography Complex peptide mixture Separated by IEX and RP Analysed by MS-MS 400 Proteins identified 2 days Protein separation Protein identification

11. Quantitation in LC based proteomics 4 Steps: 1.Label the proteins with a stable isotope 2.Mix the labeled proteins from 2 different states 3.Separate the proteins using gel or LC based techniques 4.Identify and quantify on the Mass spec Occurs on the mass spectrometer Protein quantification

12. SSP 2004 : Thiazole biosynthetic enzyme Gel based quantitation on basis of staining intensity Protein quantification

13. Step 1 Grow cells on isotope enriched media (Metabolic labelling) Or derivitise with an Isotope-Coded Affinity Tag (ICAT) 15 14 heavy (Phenotype 1) light (Phenotype 2) heavy (Phenotype 1)light (Phenotype 2) C 10 H 17 N 3 O 3 C 10 D 17 N 3 O 3 Protein quantification

14. Step 2 15N - KPGALSGLLG14N - KPGALSGLLG Phenotype 1Phenotype 2 Protein quantification

15. Step 3 15N - KPGALSGLLG 14N - KPGALSGLLG Protein quantification

16. Step 4 14N - KPGALSGLLG 15N - KPGALSGLLG N Area = 75Area = 100 N = number of N atoms present in the peptide Peptide KPGALSGLLG is More abundant in state 2 Than in state 1 Protein quantification

17. Protein separation Protein identification Protein quantification Protein generation Look at variations in expression of a certain protein under different conditions. Infer its function on basis of conditions Protein quantification: Case Study - Sulfolobus

18. Metabolic pathway/networks (re)construction Sugar metabolism Krebbs’ cycle

19. Future plans Further optimise and vary separation techniques Quantify protein expression on basis of gel staining, ICAT and metabolic labelling Metabolic pathway reconstruction in order to obtain an understanding of the central carbon metabolism of S. solfataricus Comparing different phenotypes (Carbon sources) D-Glucose (c6 sugar) D-Arabinose (c5 sugar) Tryptone (peptides) CO 2 (autotrophic)

20. Chemical/Biochemical Products Pharmaceuticals – anti-cancer, anti-HIV, antimicrobial, immuno- suppressives etc…. Enzymes/Biocatalysts (inc “extremozymes”) Processes: Combined/integrated biocatalysis-chemical catalysis (including – eg microreactors / microfluidics) Biomaterials – linkages into for example tissue engineering Biofuels – eg Hydrogen economy: H 2 generation, and sequestration/ reuse/storage of CO 2 Linkages to MBB and APS, and US metabolic engineering (Ken Reardon, Colorado State university and Tom Wood, University of Connecticut)

21. Medical Tissue Engineering –Systems Biology approach – experiments and modelling –Initial process is underway – Profs Rod Smallwood, Sheila McNeil and Jenny Southgate (York) & Dr Zimmerman (CPE) Kidney disease – with Prof El-Nahas and Dr Ong Reproductive medicine – Dr Fazeli Stem Cells (also links to MBB/BMS) –Andy Scutt – Mat Sci/Clinical Sciences

22. Systems Biology Analysis Combine time course microarray data with quantitative time course proteomic data Examine relationships between transcriptome and proteome Use of multivariate statistics to look for interactions Metabolic reconstruction – biological networks Metabolic models – for information and informing new experiments

23. Acknowledgments EPSRC HEFCE-University of Sheffield