Application of 2D in global profiling of the E.coli Proteome

Two dimensional electrophoresis can be used top study the entire protein present in the cell which can be used to identify

the specific function of each protein Related LOs: Protein property > Prior Viewing – IDD-1. Extraction of bacterial protein, IDD-11. Protein quantification, IDD-14. Isoelectric focusing, IDD-17. SDS-PAGE , IDD-19. Coomassie staining, IDD-23. DIGE gel scanning > Future Viewing – IDD-33. Western blot assay, IDD-35. Immunohistochemistry

Course Name: Application of 2D in global profiling of E.coli proteome Level(UG/PG): PG Author(s): Dinesh Raghu, Vinayak Pachapur Mentor: Dr. Sanjeeva Srivastava

Learning objectivesAfter interacting with this learning object, the

learner will be able to:1. Define to design a good experimental plan2. Identify the significance of 2D separation

technique3. Infer the function of hypothetical proteins and

the experimental proteins in the study

4. Assess the troubleshooting steps involved in the experiments.

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Master Layout

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Slide 6-11

Step 1: Audio Narration Description of the action

T1: First and second dimension separations

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Instruct the user to go through IDD-1. Extraction of bacterial protein, IDD-11. Protein quantification, IDD-14. Isoelectric focusing, IDD-17. SDS-PAGE , IDD-19. Coomassie staining, IDD-23. DIGE gel scanning, IDD-24. DIGE gel analysis. Use the pictures from previous slide to show in short the animation for all the above mentioned IDD must be a short recap of the experiment flow. Animate to start with Extraction followed by Quantification, loading of the protein sample on the strip, carrying out the focusing followed by 2-dimensional separation, scanning, staining followed by the gel analysis. Use the pictures from previous slide

How the proteome profile of a particular sample starts from Extraction followed by Quantification, loading of the protein sample on the strip, carrying out the focusing followed by 2-dimensional separation, scanning, staining followed by the gel analysis.

Step 1:

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T1: First and second dimension separations

The normal gel must be stained later rounded for each protein spotLater protein name annotation of each stop.The protein spot must be pop-out when user goes through the spots.

Step 2: Audio Narration Description of the action

T2: Mass spectrometry analysis

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Instruct the user to go through the IIDD-26. Spot picking, IDD-27. In gel digestion, IDD-30. Matrix Instrumentation and IDD-31. MALDI-TOF data analysis, IDD-39. LC-MSMS data analysis.

Instruct user to animate the screen with the figures from IDD mentioned with figure options as in next slide. Instruct the user to click on start and show like the processing sign in the screen followed with the screen from the slide:9-12. Wherever “human serum “ in the IDD is found replace all with the “E.coli” for this animation

The next part of the Proteome profiling is the identification of the protein after carrying out 1D and 2D separation. The identification part plays a major role, with more improvements in MS instrument as helped proteome analysis at rapid phase.

Step 1: T1: Title of the step, to appear as heading of the screen (if any)

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T2: Mass spectrometry analysis

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T2: Mass spectrometry analysis

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T2: Mass spectrometry analysis

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T2: Mass spectrometry analysis

Animation areaInteraction 1: slide-6: provide user the proteome

profile and let user interrupt the result.Instruction: user must be able to find the higher /low

abundance proteins, calculate their pI and molecular weight from the given profile.

Instructions/ Working area

Credits

Name of the section/stage Interactivity

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Questionnaire:APPENDIX

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Question 1:

Global proteome meansa)Entire protein content of the cellb)Specific proteins in the cellc)Only metabolically functional proteinsd)Transcription factors

Question 2

E.Coli is a

a)Gram positive bacteriab)Gram neutral bacteriac)Gram negative bacteriad)Mycobacteria

Question 3:Proteins are identified bya)Mass spectrometryb)UV –visible spectrometryc)NMRd)IR spectroscopy

Links for further readingAPPENDIX

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http://www.matrixscience.com/search_form_select.html

1. Henzel.W.J., Watanabe.C., Stults.J.T. (2003). Protein Identification: The Origins of Peptide Mass fingerprinting. J Am Soc Mass Spectrom, 14(9), pp:931-42.2. Nesvizhskii , A.I., Vitek, O., Aebersold, R. (2007). Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat.Methods., 49 (1), pp.787-97.3. Deutsch, E.W., Lam, H., Abersold, R. (2008) Data analysis and bioinformatics tools for tandem mass spectrometry in proteomics. Physiol Genomics. 33 (1), pp:18-25.4. Yates, JR., 2008. Mass Spectrometry and the Age of Proteome. J.Mass.Spec., 33(1), pp.1-19.

Books: Proteomics: A cold spring harbor laboratory course manual by Andrew J L and Joshua L, 2009.

SummaryAPPENDIX

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The application of 2D involves the global profiling of the b proteome of the organism that can used to correlate its state by validating its protein content and function of protein by 2D.