Introduction to proteomics

With the DNA sequences of more than 90 genomes completed, as well as a draft sequence of the human genome, a major challenge in modern biology is to understand the expression, function, and regulation of the entire set of proteins encoded by an organism—the aims of the new field of proteomics. This information will be invaluable for understanding how complex biological processes occur at a molecular level, how they differ in various cell types, and how they are altered in disease states. The term proteomics describes the study and characterization of a complete set of proteins present in a cell, organ, or organism at a given time.
In general, proteomic approaches can be used (a) for proteome profiling, (b) for comparative expression analysis of two or more protein samples, (c) for the localization and identification of posttranslational modifications, and (d) for the study of protein-protein interactions. The human genome harbours 26000–31000 protein-encoding genes; whereas the total number of human protein products, including splice variants and essential posttranslational modifications (PTMs), has been estimated to be close to one million. It is evident that most of the functional information on the genes resides in the proteome, which is the sum of multiple dynamic processes that include protein phosphorylation, protein trafficking, localization, and protein-protein interactions. Moreover, the proteomes of mammalian cells, tissues, and body fluids are complex and display a wide dynamic range of proteins concentration one cell can contain between one and more than 100000 copies of a single protein.
A rapidly emerging set of key technologies is making it possible to identify large numbers of proteins in a mixture or complex, to map their interactions in a cellular context, and to analyze their biological activities. Mass spectrometry has evolved into a versatile tool for examining the simultaneous expression of more than 1000 proteins and the identification and mapping of posttranslational modifications. High-throughput methods performed in an array format have enabled large-scale projects for the characterization of protein localization, protein-protein interactions, and the biochemical analysis of protein function. Finally, the plethora of data generated in the last few years has led to approaches for the integration of diverse data sets that greatly enhance our understanding of both individual protein function and elaborate biological processes.

1. Introduction to Proteomics Guided By, Dr C Amruthavalli Invited Faculty University of Mysore Presented By, SHRYLI K S YMB17118 IX Semester UNIVERSITY OF MYSORE Yuvaraja’s College (Autonomous) Mysuru - 570 005 Major seminar presented in partial fulfilment of the requirement of the Award of M.Sc. in Molecular Biology (5- Year Integrated Course) Department of Molecular Biology Yuvaraja’s College (Autonomous) (A CONSTITUENT AUTONOMOUS COLLEGE OF THE UNIVERSITY OF MYSORE) Mysore – 570 005 7 th January 2022 07-01-2022 Introduction to Proteomics_Shryli K S 1

2. Contents Introduction History Objective How are the proteins studied? Protein detection with antibodies Antibody-free protein detection High throughput protein profiling Protein Databases Journals Summary Conclusion References Acknowledgement 07-01-2022 Introduction to Proteomics_Shryli K S 2 Fig 01: 3D structure of Sharpin protein.

3. An apple a day keeps the doctor away! Energy: 218 kJ (52 kcal) Calories: 52. Water: 86% Protein: 0.27 grams. Carbs: 13.81 grams. Sugar: 10.4 grams. Fiber: 2.4 grams. Fat: 0.17 grams. Malus domestica Malus domestica Double Bond Reductase B iphenyl synthase Chalcone synthase ACC synthase A pple tyrosinase Malus domestica Double Bond Reductase PDB ID: 6YUX 07-01-2022 Introduction to Proteomics_Shryli K S 3 Fig 02: Proteins in apple.

4. Introduction 07-01-2022 Introduction to Proteomics_Shryli K S 4 “T he study and characterization of complete set of proteins present in a cell, organ, or organism at a given time.” P roteome profiling. Comparative expression analysis of two or more protein samples. The localization and identification of posttranslational modifications. T he study of protein–protein interactions. 26,000 – 31,000 Genes 1 million Proteins!!! Splice variants & Post translational Modifications

5. History 07-01-2022 Introduction to Proteomics_Shryli K S 5 1975 Escherichia coli (2-D Gel) PROTEIN GENOME + PROTEOME (1994) Fig 03: E. coli Fig 04: Prof. Marc Wilkins Fig 05: Macquarie University, Sydney. Fig 06: Dr Karthik S Kamath Senior Analytical Technician - Mass Spectrometry

6. Objectives of Proteomics 07-01-2022 Introduction to Proteomics_Shryli K S 6 “A detailed proteomic study facilitates or enables the scientist to get a profound insight about the biological and physiological status of the cell, which is not so vivid and detailed in the case of genomic studies” P rotein–protein interactions Protein-ligand interactions Fig 07: P rotein–ligand interactions . Fig 08: P rotein–nucleic acid interactions . Protein nucleic acid interaction Structure Expression Function

7. How are the Proteins Studied? 07-01-2022 Introduction to Proteomics_Shryli K S 7 Fig 09: 1°, 2° and 3° structures of the protein Albumin

8. Protein Detection with Antibodies 07-01-2022 Introduction to Proteomics_Shryli K S 8 Fig 10: The western blotting technique. The western blotting technique. 1979 Fig 11: Sir Walter Neal Burnette

9. 07-01-2022 Introduction to Proteomics_Shryli K S 9 The  enzyme-linked immunosorbent assay. Fig 13: From left to right, Dr. Eva Engvall (Sweden), Dr. Anton Schuurs (The Netherlands), Dr. Peter Perlmann (Sweden), Dr. Bauke van Weemen (The Netherlands), and Prof. Johannes Büttner (Germany), President of the German Society of Clinical Chemistry . 1971 Fig 12: The ELISA technique.

10. 07-01-2022 Introduction to Proteomics_Shryli K S 10 Immunohistochemistry. 1942 Fig 15: Prof. Albert H Coons. Fig 16: The IHC technique.

11. 07-01-2022 Introduction to Proteomics_Shryli K S 11 Edman Degradation Method. 1967 Fig 17: The Edman Degradation Method. Fig 18: Pehr Victor Edman Antibody-free protein detection

12. 07-01-2022 Introduction to Proteomics_Shryli K S 12 Fig 19: X-Ray crystallography protein. X-Ray crystallography protein. 1934 Fig 20: J ohn Desmond Bernal and Dorothy Crowfoot Hodgkin.

13. 07-01-2022 Introduction to Proteomics_Shryli K S 13 Fig 22: Prof. Dr. P. H. O'Farrell, Walter Sarstedt , Prof. Dr. Dr. J. Klose. 1975 2 Dimensional Differential Gel Electrophoresis. Fig 21: 2 Dimensional Differential Gel Electrophoresis technique.

14. 07-01-2022 Introduction to Proteomics_Shryli K S 14 Fig 24: Nobel Laurate Koichi Tanaka 198 7 Mass spectrometry. Fig 23: Mass spectrometry technique.

15. Protein Databases 07-01-2022 Introduction to Proteomics_Shryli K S 15 PROTEIN SEQUENCES DATABASE USES DisProt Database of experimental evidences of disorder in proteins InterPro Classifies proteins into families and predicts the presence of domains and sites MobiDB Database of intrinsic protein disorder annotation neXtProt A human protein-centric knowledge resource Pfam Protein families database of alignments and HMMs  PRINTS A compendium of protein fingerprints PROSITE Database of  protein families  and  domains Protein Information Resource Protein Information SUPERFAMILY Library of HMMs representing superfamilies and database of (superfamily and family) annotations for all completely sequenced organisms Swis-Prot Protein knowledgebase NCBI Protein sequence and knowledgebase Table 01: List of databases for protein sequences. “A  protein  database is a collection of data that has been constructed from physical, chemical and biological information on sequence,  domain  structure, function, three‐dimensional structure and protein‐protein interactions.”

16. 07-01-2022 Introduction to Proteomics_Shryli K S 16 PROTEIN MODELS DATABASE USES ModBase Database of comparative protein structure models Similarity Matrix of Proteins (SIMAP) Database of protein similarities computed using  FASTA Swiss-model Server and repository for protein structure models AAindex Database of amino acid indices, amino acid mutation matrices, and pair-wise contact potentials PROTEIN EXPRESSION Human Protein Atlas Aims at mapping all the human proteins in cells, tissues and organs PROTEIN STRUCTURE Protein Data Bank (PDB) Structural Classification of Proteins (SCOP) CATH INTERACTIONS BioGRID RNA-binding protein database Database of Interacting Proteins IntAct Table 02: List of databases for protein models, expression, structure and interactions.

17. 07-01-2022 Introduction to Proteomics_Shryli K S 17 Applications

18. Journals 07-01-2022 Introduction to Proteomics_Shryli K S 18 Fig 25: Proteomics Journals .

19. Summary 07-01-2022 Introduction to Proteomics_Shryli K S 19 Classification Procedure Techniques Data Analyzers PDB Fig: An overview of proteomic strategies. Source: Chandramouli and Qian. 2009

20. Conclusion 07-01-2022 Introduction to Proteomics_Shryli K S 20 2021 : David Julius  and  Ardem Patapoutian  “for their discoveries of receptors for temperature and touch ”. 2018 :James P. Allison  and  Tasuku Honjo  “for their discovery of cancer therapy by inhibition of negative immune regulation” 2016 : Yoshinori Ohsumi  “for his discoveries of mechanisms for autophagy” 2009 : Elizabeth H. Blackburn ,  Carol W. Greider  and  Jack W. Szostak  “for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase” 2004 : Richard Axel  and  Linda B. Buck  “for their discoveries of odorant receptors and the organization of the olfactory system” 2003 : Paul C. Lauterbur  and  Sir Peter Mansfield  “for their discoveries concerning magnetic resonance imaging” 1992 : Edmond H. Fischer  and  Edwin G. Krebs  “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism” 1988 : Sir James W. Black ,  Gertrude B. Elion  and  George H. Hitchings  “for their discoveries of important principles for drug treatment”

21. References 07-01-2022 Introduction to Proteomics_Shryli K S 21 Lynn B Jorde et.al. (2005), Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics, Wiley, 4046 pgs. Michael J. Dunn (2000), From Genome to Proteome: Advances in the Practice and Application of Proteomics, Wiley,551 ps. Aslam, B., Basit, M., Nisar, M. A., Khurshid, M., & Rasool, M. H. (2017). Proteomics: Technologies and their applications. In Journal of Chromatographic Science (Vol. 55, Issue 2). https://doi.org/10.1093/chromsci/bmw167 Chandramouli , K., & Qian, P.-Y. (2009). Proteomics: Challenges, Techniques and Possibilities to Overcome Biological Sample Complexity. Human Genomics and Proteomics , 1 (1). https://doi.org/10.4061/2009/239204 Coon, J. J., Syka , J. E. P., Shabanowitz , J., & Hunt, D. F. (2005). Tandem mass spectrometry for peptide and protein sequence analysis. In BioTechniques (Vol. 38, Issue 4). https://doi.org/10.2144/05384TE01 Gonçalves-Maia, R. (2012). From X-rays to biomolecular structure: D. Hodgkin, R. Franklin and A. Yonath . Revista Virtual de Quimica , 4 (6). https://doi.org/10.5935/1984-6835.20120059 Lequin , R. M. (2005). Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical Chemistry , 51 (12). https://doi.org/10.1373/clinchem.2005.051532

22. 07-01-2022 Introduction to Proteomics_Shryli K S 22 Lundström, S. L., Zhang, B., Rutishauser , D., Aarsland , D., & Zubarev, R. A. (2017). SpotLight Proteomics: Uncovering the hidden blood proteome improves diagnostic power of proteomics. Scientific Reports , 7 . https://doi.org/10.1038/srep41929 Patterson, S. D., & Aebersold , R. H. (2003). Proteomics: The first decade and beyond. In Nature Genetics (Vol. 33, Issue 3S). https://doi.org/10.1038/ng1106 Popescu, S. C., Popescu, G. v., Bachan , S., Zhang, Z., Seay, M., Gerstein, M., Snyder, M., & Dinesh-Kumar, S. P. (2007). Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays. Proceedings of the National Academy of Sciences of the United States of America , 104 (11). https://doi.org/10.1073/pnas.0611615104 Shah, T. R., & Misra , A. (2011). Proteomics. Challenges in Delivery of Therapeutic Genomics and Proteomics , 387–427. https://doi.org/10.1016/B978-0-12-384964-9.00008-6 Zhu, H., Bilgin , M., & Snyder, M. (2003). Proteomics. In Annual Review of Biochemistry (Vol. 72). https://doi.org/10.1146/annurev.biochem.72.121801.161511 Kwon M., Cho S.Y., Paik Y. (2005) Protein Databases. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Springer, Berlin, Heidelberg . https://doi.org/10.1007/3-540-29623-9_3520

23. Acknowledgements 07-01-2022 Introduction to Proteomics_Shryli K S 23 I would like to thank the department of Molecular Biology for providing me this opportunity to present my seminar, I thank my guide Dr C Amruthavalli for her guidance. Thank you all for your patience.

24. 07-01-2022 Introduction to Proteomics_Shryli K S 24 Gregory Stock is a biophysicist, best-selling author, biotech entrepreneur, and the former director of the Program on Medicine, Technology and Society at UCLA’s School of Medicine.

25. 07-01-2022 Introduction to Proteomics_Shryli K S 25 Fig 24: The first paper published on the protein microarray technique by Popescu SC, Popescu GV, Bachan S, Zhang Z, Seay M, Gerstein M, Snyder M, Dinesh‐Kumar SP. 2007 P rotein Microarray Fig 23: P rotein Microarray technique.

26. 07-01-2022 Introduction to Proteomics_Shryli K S 26 Fig 26: Edward Mills Purcell and Felix Bloch shared the 1952 Nobel Prize in Physics for their discoveries. The Purcell group at Harvard University and the Bloch group at Stanford University independently developed NMR spectroscopy in the late 1940s and early 1950s Nuclear Magnetic Resonance Fig 25: Nuclear Magnetic Resonance