Introduction to Proteomics Susan Liddell University of Nottingham susan.liddell@nottingham.ac.uk

1. Introduction to Proteomics Susan Liddell University of Nottingham susan.liddell@nottingham.ac.uk PGT short course May 2012 UoN Graduate School Course Post-genomics and bio-informatics

2. Sutton Bonington Proteomics labsDivision of Animal Sciences – South labSusan Liddell, Ken Davies • Supports proteomics studies & collaborative projects • gel electrophoresis (mainly 2D) • protein identification via tandem MS • Wide variety of types of projects and organisms • including some species with unreported genome sequences human cow horse fungi bacteria archaea plants Dr Ken Davis

3. Overview • what is proteomics? • why study the proteome • proteomic strategies • the 2D gel standard workflow • 2D DiGE • High throughput LC-MSMS

4. Proteome “the PROTein complement of a genOME” Wasinger et al 1995 Electrophoresis: 16:1090 Proteomics “...the identification of all the proteins encoded in the human genome....” including modification, quantification, localisation and functional analysis for every cell type Human Proteome Organisation (www.hupo.org)

5. Proteomics study of proteins and protein function usually on a genome wide scale Proteomics preceded genomics Human Protein Index N & L Anderson 1982

6. Aims of Proteomics • Global analysis of complex samples • A fundamental understanding of biological processes and mechanisms • Find changes in protein expression (biomarkers) in different biological situations (disease) • Aid in development of therapeutic agents/drugs

7. Why analyse the proteome? Genome considerations sequence alone does not reveal biological function Arabidopsis genome annotation functional characterisation 26% molecular function unknown Functional Annotation of the Arabidopsis Genome Using Controlled Vocabularies Plant Physiology (2004) Vol.135, p745

8. Why analyse the proteome? Genome considerations • one gene can code for more than one protein • gene rearrangements • RNA splicing • unlike the genome, the proteome is highly dynamic • varies from tissue to tissue • between different cell types • according to developmental stage • environment (e.g. disease)

9. Why analyse the proteome? Transcript considerations • poor correlation between mRNA and protein expression levels • Gygi et al 1999 Correlation between protein and mRNA abundance in yeast. Mol.Cell.Biol. 19:1720 • Anderson and Seilhamer. 1997 A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18:533 • Ingolia et al 2009 Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924):218-23 • each transcript can give rise to several protein isoforms via post translational processing (>300 PTMs)

10. Common covalent modifications affecting protein activity Biochemistry. Berg, Tymoczko, Stryer, Clarke

11. PROTEOMICS • proteins are the main biological effector molecules • not just identifying novel genes, now determining the function of gene products • analysis of protein products complements genomics & transcriptomics

12. “At the end of the day, proteins, not genes, are the business end of biology.”

13. Targeted Proteomics Global Proteomics identify all proteins present • quantitative changes : abundance • qualitative changes : PTMs • subcellular compartments : nuclei, membranes • functional: complexes of interacting proteins

14. There are many Proteomic Approaches using many different technologies GelsProteins1D/2D gelsstains/labels Liquid ChromatographyPeptides/Proteins1D/2DLabels/label free Protein ChipsProtein arrays on slides Mass Spectrometry

15. The Nobel Prize in Chemistry 2002 "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules" • Electrospray ionization (ESI) • John B Fenn • Matrix-assisted laser desorption/ionization (MALDI) • Koichi Tanaka

16. Applications of mass spectrometry in protein analysis include Protein identification peptide mass fingerprinting Tandem MS de novo sequence Recombinant protein evaluation confirm identity engineered mutations, sequence changes cleavages or other modifications assess homogeneity Identification of modifications acetylation oxidation glycosylation phosphorylation ….anything that causes a change in mass….

17. Proteomic Workflow 2D gel/MS Proteinseparation Analysis and protein spot selection Processing and digestion to peptides Mass spectrometric analysis Database interrogation Protein identification

18. kDa Protein separation2-dimensional gel electrophoresis pI 1st dimension Separation by charge (isoelectric focussing) pH 3 pH 10 2nd dimension Separation by molecular weight (SDS-PAGE)

19. 2D gel electrophoresis equipment1st dimension IEF various lengths 5 - 24 cm wide range pH 3-11 narrow/zoom range pH 4-5 loading methods in-gel rehydration cup, paper bridge

20. 2-D gel electrophoresis equipment 2nd dimension SDS-PAGE various lengths linear / gradient reducing / non-reducing Multi-gel runners increase reproducibility increase throughput

21. Protein detection and image capture post-gel staining colloidal coomassie blue silver SYPRO ruby, Deep Purple, Flamingo pre-gel sample labelling 35S-methionine Cy3, Cy5, Cy2 (DiGE) Pro-Q Diamond – phosphoproteins Pro-Q Emerald – glycoproteins Pro-Q Amber – transmembrane proteins (1D gels)

22. Example 2D gelE. coli cell extract 250 150 100 75 Soo Jin Saa 100 µg pH 4-7 IPG strip 12.5% PAGE Silver stained 50 37 25 20

23. Comparison of gel stains SYPRO ruby ~ 1 ng/mm2 Silver 0.5 ng/mm2 Colloidal Coomassie Blue 10-50 ng/mm2

24. Special cases Bacteria -high nucleic acid: protein ratio -use nucleic acid removal techniques Yeast/fungi -tough cell walls require vigorous disruption to lyse -protease activity high Cultured cells -salt (especially phosphate ions) from medium -wash in salt free buffer / osmoticum Plant tissues -dilute source of protein -precipitation is usually used -protease activity is high -reductants/inhibitors to prevent phenolic modification

25. Proteomic Workflow 2D gel/MS Proteinseparation Analysis and protein spot selection Processing and digestion to peptides Mass spectrometric analysis Database interrogation Protein identification

26. Analysis and spot selection Find differences in spot patterns (protein expression changes) between samples using image analysis software Image analysis software PDQuest (BioRad) DeCyder (GE Healthcare) Same Spots (Nonlinear Dynamics) Image capture Spot detection Spot matching across gel set Statistical evaluations

27. Hereditary bovine dilated cardiomyopathy:11 proteins increased in abundance Weekes et al (1999)Electrophoresis 20:898

28. Proteomic Workflow 2D gel/MS Proteinseparation Analysis and protein spot selection Processing and digestion to peptides Mass spectrometric analysis Database interrogation Protein identification

29. Gel spot excision and processing Pick individual spots into 96-well microtitre plates Destain Digest (trypsin) Peptide extraction

30. Proteomic Workflow 2D gel/MS Proteinseparation Analysis and protein spot selection Processing and digestion to peptides Mass spectrometric analysis Database interrogation Protein identification

31. Identify proteins using Mass Spectrometry MALDI-ToF Q-ToF2 (plus capillary/nano flow HPLC)

32. Investigation of proteins involved in ovarian folliculogenesis Ken Davis, Jacqueline Cameron, Susan Liddell & Bob Webb School of Biosciences

33. Ovulation 1 to 2 Follicle number 2 to 6 15 to 20 Dominance Dominance Selection Dominance Selection Selection Selection phase phase 18 15 9 12 6 0 3 9 12 21 Days of oestrous cycle Waves of follicular growth and development in the cow early stage“dominant” follicles oocytes with a lower fertilisation rate True dominant follicles healthier fertilisable oocytes

34. Granulosa cells surround the oocyte GCs provide instructions directing oocyte development oocyte granulosa cells

35. ovarian follicle granulosa cell proteins Prepared GC extracts from each stage Compare profiles Identify proteins that differ Sets of analytical gels run Samespots™ software indicates we need more gels for acceptable statistics Project ongoing ..... Proteins that determine which follicle becomes the dominant, mature follicle that can be fertilized determine the quality of the oocyte

36. Establish proteome maps of GC proteins Reference profile to support differential analyses projects So far identifying proteins of a fairly abundant “housekeeping” nature How to find regulator proteins – of lower abundance? GRP94 GLUCOSIDASE II ATPase HSP70 CALREGULIN ALBUMIN TUBULIN HSP60 PROTEIN DISULPHIDE ISOMERASES ENOLASE ACTINS ALDEHYDE REDUCTASE ANNEXIN PHOSPHOGLYCERATE MUTASE UBIQUITIN PEROREXOXIN HAEMOGLOBIN

37. Limitation of Proteomic Technologies Dynamic range Don’t see the lower abundance proteins in complex mixtures

38. Proteins measured clinically in plasma span > 10 orders of magnitude in abundance Anderson NL, Anderson NG The human plasma proteome: history, character, and diagnostic prospects Molecular and Cellular Proteomics 2002 1:845-867

39. 1010 Really Is Wide Dynamic Range(Here on a linear scale)

40. How to overcome the dynamic range and detect proteins of lower abundance? 2D gels only ~2-3 orders of proteins detected only the most abundant proteins Mass spectrometers detection range of ~ 3 (to 5) orders Reduce the complexity and dynamic range Fractionation techniques include remove abundant proteins different cellular compartments differential protein solubility sequential chromatography (2D, 3D) affinity purification

41. Fractionation : Remove abundant proteins12 proteins in plasma comprise ~ 96% of the protein mass Figure courtesy of Beckman Coulter

42. Immunodepletion of 6 high abundance proteins from human serum M 1 2 3 1 - crude serum 2 – flow through fraction depleted of high abundance proteins 3 - bound fraction Figure courtesy Agilent Technologies

43. RuBiSCo immunodepletion • the biggest single obstacle in plant proteomics • ribulose-1,5-bisphosphate carboxylase oxygenase • enzyme catalyses the first major step of carbon fixation • the energy supplied by photosynthesis is used to convert carbon dioxide into available food • ~ 40%-60% of the total protein in green plant tissues • the most abundant protein in plants • the most abundant protein on Earth

44. RuBiSCo immunodepletion tool IgY antibodies raised against purified RuBiSCo from spinach cross species Petunia leaf Arabidopsis leaf Total Depleted Total Depleted 250 150 100 75 50 Large Sub-Unit 37 25 20 15/10 Small Sub-Unit Susan Liddell, unpublished

45. Reduce the complexity and dynamic range Fractionation techniques include remove abundant proteins different cellular compartments differential protein solubility sequential chromatography (2D, 3D) affinity purification

46. Fractionation: Different cellular compartments example of a subproteome experiment Investigation of proteins involved in nuclear transfer Jacqueline Cameron, Susan Liddell & Keith Campbell School of Biosciences

47. Nuclear transfer spindle Enucleation MII oocyte Inject new, somatic nucleus DC electric pulse Development Transfer to surrogate Gestation

48. spindle The meiotic spindle is removed from oocyte Does this also deplete associated proteins that are required for subsequent development and cell cycle control? Identification of such proteins would provide deeper understanding of the process and allow controlled replenishment to improve the outcome of NT

49. DIfference Gel Electrophoresis (DIGE) Sample 1 Cy3 Sample 2 Cy5 Label samples Mix samples run on ONE gel Cy3/Cy5 Scan gel capture Cy3, then Cy 5 overlay the images Analyse Unlu M, Morgan ME, Minden JS Electrophoresis1997 Difference gel electrophoresis: a single gel method for detecting changes in protein extracts Figure adapted from Cambridge Centre for Proteomics

50. DeCyder analysis – Example protein spot Spot 1007 increased, volume ratio 3.20