Master’s degree in Proteomics & Bioinformatics January 2008 Master’s report November 2006 –April 2007 Characterisation of human cerebrospinal fluid (CSF) after Tandem Mass Tag (TMT0) labelling Virginie LICKER Supervision: Vaksha Patel & Dr. Malcolm Ward, Proteome Sciences Geneva University coordinator: Dr. Jean-Charles Sanchez, BPRG Proteome Sciences Plc Laboratory, London Institute of Psychiatry King’s College London De Crespigny Park London SE5 8AF United Kingdom Contact: [email protected] A la mémoire d’Alexis Etudiant MPB 2005-2007 Son amitié, sa générosité, sa sincérité, son sens de l’humour et sa bonne humeur communicative, resteront des souvenirs impérissables de ces années communes d’Université. 2 Acknowledgements I would like to express my gratitude to Dr. Malcolm Ward, head scientist of Proteome Sciences London’s research team, for welcoming me and allowing me to complete my internship in Proteome Sciences laboratory. I would like to thank particularly Vaksha Patel my supervisor and Dr. Helen Byers, for guiding me throughout my first labwork experiments as well as for their scientific advice. Many thanks to all my other lab’s mates, James, Annette, Darragh, Abdul, “Maxton” and the King’s academics, Andy, Mirreia and Steve for their care, kindness, help and for making my everyday life in the lab’ fun. I would like to thank Dr. Jean-Charles Sanchez for allowing me to continue the reflexion initiated in London on the Geneva Proteomics plateform. I am grateful for his patience and for trusting in my capabilities, as well as for his encouragements and communication skills. I would also like to thank Loic Dayon, for letting me benefit of his knowledge on TMT and other techniques through scientific discussions and lab’ experiments. Thanks to my MPB classmates (Yann, Harris, Ilona, Bea, Nath, Greg & Cie) and the members of the BPRG for stimulating me to write up my report! Last but not least, I would like to thank my friends, especially Dany and Alain, as well as my family for their flawless support. 3 Characterisation of human cerebrospinal fluid (CSF) after Tandem Mass Tag (TMT0) labeling A. ABSTRACT…………………………………………………………………...p.7 B. INTRODUCTION…………………………………………………………….p.8-17 1. Introduction to proteomics p.8 2. Sample preparation for mass spectrometry: Chromatography p.9-11 2.1 Principles and Instrumentation 2.2 Reversed Phase and Strong Cation Exchange characteristics 2.3 Workflows 3. Mass Spectrometry p.11-13 3.1 Principles and Instrumentation (Maldi-TOF and Q-TOF) 3.2 Protein identification 3.3 Database search and interpretation 4. Quantitative Proteomics p.13-15 5. Biomarker discovery in the Cerebrospinal Fluid (CSF) p.15-16 6. Description of the project p.16 C. MATERIAL AND METHODS……………………………………………....p.17-23 1. Reagents p.17 2. Samples p.17 3. Determination of total protein concentration: Bradford assay p.17-18 4. Gel Electrophoresis and Staining p.18 5. Digestion p.18-19 5.1 In-solution digestion 5.1.1. Standard 5.1.2 TMT protocol 5.2 In-gel digestion: TMT protocol 6. Labeling using TMT0 reagent p.19 7. Chromatography p.19-21 7.1 Manual SCX 7.2 HPLC 7.2.1 RPC18-SCX in series 7.2.2. a. RPC18 b. SCX 8. Sample preparation for mass spectrometry: Zip-tipping with C18 p.21 9. Mass Spectrometry p.22-23 9.1. Protein identification by Peptide Mass Fingerprinting (PMF) 9.2.1. Sample preparation 9.2.2. Data acquisition: MALDI-TOF Voyager DE Pro 4 9.2.3. Data analysis: PMF 9.3. Protein identification by tandem Mass Spectrometry (MS/MS) 9.3.1. Sample preparation 9.3.2. Data acquisition: ESI Q-TOF Micro 9.3.3. Data analysis D. RESULTS & DISCUSSION…………………………………………….……p.24-42 1. Sample preparation and pre-fractionation for shotgun proteomics using isobaric reagent TMT p.24-31 1.1. Evaluation of TMT protocols using BSA a) Digestion protocols: “in solution” and “in gel” digestion b) Buffers: TEAB versus Borate buffer c) Labeling “efficiency” 1.2. Optimization of the purification and fractionation of TMT0 labeled peptides on HPLC using BSA a) Columns configuration b) Buffer composition c) Sample preparation - RP C18 - SCX d) Method 2. Mass spectrometry p.32-36 2.1. MS and MS/MS data for the identification and quantification of proteins 2.1.1. MALDI-TOF analysis 2.1.1.1. Sample preparation: methods and matrix 2.1.1.2. Peptide representation 2.1.1.3. Limits of detection (gel, peptide standard) 2.1.1.4 Applications: - Protein identification: PMF - Quality control 2.1.2. ESI-Q-TOF analysis 2.1.2.1. Sample preparation: zip-tipping or not? 2.1.2.2. Protein identification & quantification 3. Protein profiling of CSF after isobaric TMT labeling p.37-41 3.1. Sample preparation 3.1.1. Determination of total protein amounts 3.1.2. Workflow 3.1.3. Influence of buffer pH on SCX fractionation 5 3.2. Protein analysis 3.2.1. Protein identification 3.2.2. Protein quantification 3.2.3. Biological signification 3.3.1. Method improvements E. CONCLUSION………………………………………………………………...p.42-43 F. TABLES………………………………………………………………………..p.44-46 G. REFERENCES………………………………………………………………...p.47-48 6 A. ABSTRACT Clinical diagnosis of neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease is still unsatisfactory and requires sensitive and specific biomarkers for their detection, prevention and treatment. Extensive characterisation of cerebrospinal fluid (CSF) can contribute to a better understanding of underlying pathogenetic neurodegenerative mechanisms and provide a valuable source for biomarker discovery, due to its brain proximity and privileged connection to blood. The analysis of the CSF proteome is challenging because of its diversity and high dynamic range that requires very robust and sensitive quantitative proteomics plateforms to detect down to femtomole protein levels. In this project, a shotgun proteomics approach using the isobaric tandem mass tags TMT0 was employed to label undepleted human CSF, followed by an off-line 2D-LC and identification of proteins with tandem mass spectrometry (MS/MS). The optimised protocol allowed the characterisation of 73 proteins of which 67 were labeled. Among them, brain specific proteins and other documented potential biomarkers were found. These results demonstrated the applicability of the established protocol and suggested that multiplex quantitative proteomics methods using the TMT technology can be applied to CSF for the detection of neurodegenerative biomarkers in up to six samples simultaneously. 7 B. INTRODUCTION 1. Introduction to proteomics The term “proteomics” refers to the systematic study of all proteins present in a cell or tissue at a given time, to describe their structure, function and expression in various biological systems1. As proteins are involved in almost all biological activities, their identification and the determination of their covalent structures have been central to life sciences, providing a window into complex cellular regulatory networks2. Before the genomics revolution, chemical methods such as the stepwise Edman N- terminal degradation were used to determine the sequence of single, highly purified proteins3. With the emergence of genomics, time consuming methods were replaced by large scale protein identifications using the correlation of mass-spectrometric measurements with newly available translated gene sequence databases. As rapid identification of proteins can now be achieved for most of the species, current proteomic studies were extended to the systematic determination of diverse properties of proteins including their sequence, quantity, post-translational modifications, protein-protein interactions and structure4. The analysis of the full proteome remains challenging because of its size and unknown complexity. The number of genes in a species is not representative of the number of proteins that is, in comparison, greatly increased as a result of alternative splicing, RNA editing and post-translational modifications for example. Moreover, the range of protein concentrations generally exceeds the dynamic range of any single analytical method. High-throughput proteomic analysis workflows generally consist in three stages: proteins separation coupled with mass spectrometry analysis and finally identification with the help of bioinformatics tools. The two most common approaches in proteomics (Figure 1) rely either on the fractionation at the protein level by one or two dimensional gel electrophoresis (“gel based”) or on the separation at the peptide level (“gel free” or “shotgun proteomics” ) by one or two dimensional liquid chromatographic (2D-LC) methods. Both techniques involve the protein digestion with a site-specific enzymatic protease (i.e trypsin) into peptides prior to mass spectrometry, and are often referred as “bottom up” approaches5. The shotgun strategy has allowed the high-throughput identification of thousands of proteins from highly complex mixtures such as cell lysates6. 7 Figure 1. Proteomics analysis by (a) gel-based and (b) gel-free approaches (Fournier et al, 2007) . In bottom-up proteomics, the analytes introduced into the mass spectrometer are peptides generated by enzymatic cleavage of one or many proteins. A. Gel approach: A protein mixture is separated by two-dimensional electrophoresis, first by isoelectric focusing then by SDS-PAGE. After visualization, protein spots are excised from the gel, digested, and analyzed by mass spectrometry for identification by database searching. B. Gel-free approach (shotgun proteomics): A protein mixture is directly digested by a specific enzyme into a peptide mixture separated by multidimensional