lecture-01-ppt-v2 · 2017. 8. 4. · scott d. patterson & ruedi h. aebersold. 12/6/12 11 iit...
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Dr. Sanjeeva Srivastava IIT Bombay
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Genome
Transcriptome
Proteome
Splicing Post-translational modification (PTM)
IIT Bombay Proteomics Course NPTEL 2
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Protein Extraction
Protein Separation
Protein Identification
Protein Characterization
IIT Bombay Proteomics Course NPTEL 3
Proteomics
IIT Bombay Proteomics Course NPTEL 4 Curr Opin Chem Biol 2005, 9, 14-9
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Proteomics 2D Gel Electrophoresis
Mass spectrometry
Nucleotide sequencing ESTs/Genome scale
Algorithms Genetic approaches
Chip based approaches
1975 1996 1994 1990 2002
time
Complex mixture analysis (LC MS/MS)
IIT Bombay Proteomics Course NPTEL 5 Nature genetics 2003, 33, 311-323
IIT Bombay Proteomics Course NPTEL
• Protein chemistry to proteomics
• Diversification of proteomics technologies
• Advent of novel technology platforms
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IIT Bombay Proteomics Course NPTEL 7
IIT Bombay Proteomics Course NPTEL
• Gene expression regulated by post-transcriptional and post-translational modifications
• Compared to genome of an organism proteome presents further complexity
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IIT Bombay Proteomics Course NPTEL 9
IIT Bombay Proteomics Course NPTEL
• Orderly and unidirectional flow of information encoded in base sequences of cells, DNA to RNA and protein
• Understanding biological system is a challenging task
• Omics approaches are promising
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IIT Bombay Proteomics Course NPTEL 11
IIT Bombay Proteomics Course NPTEL 12
IPG strip
2-D ELECTROPHORESIS (2-DE)
SDS-PAGE
DIFFERENCE GEL ELECTROPHORESIS
(DIGE)
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IIT Bombay Proteomics Course NPTEL 13
Dass et al. DOI: 10.1002/9780470118498.ch3
IIT Bombay Proteomics Course NPTEL 14
Ioniza'on Mass Analyzer Detec'on
Detects ions
Forms ions
(charged molecules)
Sort Ions by Mass (m/z)
Data Processing
Rela=ve
Abu
ndance
m/z 1000 2000
Mass Spectrum
Data System
Ion Source
Ion Detector
Sample Inlet
Mass Analyzer
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IIT Bombay Proteomics Course NPTEL 15
Ramachandran et al. 2005. FEBS J; Chandra et al. 2010. Proteomics
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Functionalized array surface
Heterologous host (e.g. E. coli)
Gene of interest
Gene insert
Protein expression
Protein purification Expression
vector
Chromatography
Protein purification SDS-PAGE
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IIT Bombay Proteomics Course NPTEL 17
Bound antibodies Flow cell system
Gold film
Change in angle of reflection
Free antigen
Glass slide
Antigen-Antibody complex
Prism
IIT Bombay Proteomics Course NPTEL 18 Ray et al. 2010. Proteomics
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IIT Bombay Proteomics Course NPTEL
• Unraveling structural and functional details of proteins at the proteome level is a daunting task
• Proteomics has quickly evolved to become an integral aspect of human biology and medicine
• Proteomics has advanced rapidly; however, many experimental and computational challenges still exist
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IIT Bombay Proteomics Course NPTEL
• Introduction to Proteomics: Tools for the New Biology, D.C. Liebler, Humana Press, 2002.
• Principles of Proteomics, R.M.Twyman, BiosScientific Pub.,2004.
• Proteomics for Biological Discovery, T.D.Veenstra, J.R.YatesIII, John- Wiley & Sons, Hoboken, New Jersey, USA; 2006.
• Protein Biochemistry and Proteomics (The Experimenter Series), R. Hubert, Academic Press, 2006.
• Proteomics in Practice: A Guide to Successful Experimental Design, R. Westermeier, T. Naven, H-R. Höpker, Wiley-VCH, 2008.
• Curr Opin Chem Biol. 2005 Feb;9(1):14-9. Protein microarrays as tools for functional proteomics. LaBaer J, Ramachandran N.
• Nature genetics 2003, 33, 311-323. Proteomics: the first decade and beyond - Nature Genetics. Scott D. Patterson & Ruedi H. Aebersold.
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IIT Bombay Proteomics Course NPTEL
• Ray et al., 2010. Label-free detection techniques for protein microarrays: Prospects, merits and challenges, Proteomics. Volume 10, Issue 4, pages 731–748, No. 4.
• Ray et al. 2012. Virtual Labs in proteomics: New E-learning tools. J of Proteomics. Volume 75, Issue 9, 17 May 2012, Pages 2515–2525
• Chandra et al. 2010. Cell-free synthesis-based protein microarrays and their applications. Proteomics. Volume 10, Issue 4, pages 717–730, No. 4 February 2010.
• Ramachandran et al. 2005. Emerging tools for real-time label-free detection of interactions on functional protein microarrays. FEBS J. Volume 272, Issue 21, pages 5412–5425, November 2005.
• Qiang Tian1, W. Andy Tao. Chemical and Functional Genomic Approaches to Stem Cell Biology and Regenerative Medicine. Chapter 10. Proteomics in Stem Cells. ISBN: 9780470041468
IIT Bombay Proteomics Course NPTEL
• Dass C. Chapter 3. Mass Analysis and Ion Detection. Fundamentals of Contemporary Mass Spectrometry. DOI: 10.1002/9780470118498.ch3.
• Arthur H. J Clin Invest. Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions. 2002;110(9):1221–1232. doi:10.1172/JCI16781. Volume 110, Issue 9 (November 1, 2002).
• Ray et al. 2010. Nanotechniques in proteomics: Current status, promises and challenges. J of Proteomics. Volume 25, Issue 11, 15 July 2010, Pages 2389–2401.
• Roden LD. 1996. Global Analysis of a Macromolecular Interaction Measured on BIAcore. Biochemical and Biophysical Research Communications. Volume 225, Issue 3, 23 August 1996, Pages 1073–1077
• Dhingra et al. 2005. New frontiers in proteomics research: A perspective. International Journal of Pharmaceutics. Volume 299, Issues 1–2, 11 August 2005, Pages 1–18.
• Homola et al. Present and future of surface plasmon resonance biosensors. Analytical and Bioanalytical Chemistry. October 2003, Volume 377, Issue 3, pp 528-539
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IIT Bombay Proteomics Course NPTEL
• My mentors Profs. Nat Kav and Joshua LaBaer
• All the guest professors and application experts for stimulating discussion
• All students and support staff (Ms. Harini S., Ms. Renisa D., Dr. Parvez S., Dr. Jaya V., Mr. Saurabh Y., Mr. Khaja S., Mr. Sandipan R., Mr. Jaipal PR, Mr. Karthik K., Ms. Rekha J.) for their help and contributions in NPTEL proteomics course
• CDEEP team (Mr. Arun K., Mr. Tushar D., Ms. Sangeeta S.)