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

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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
• gt 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
• gt 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

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Learning objectives
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• After interacting with this learning object, the
  learner will be able to
• Define to design a good experimental plan
• Identify the significance of 2D separation
  technique
• Infer the function of hypothetical proteins and
  the experimental proteins in the study
• Assess the troubleshooting steps involved in the
  experiments.

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Master Layout
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2
Slide 4-5
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4
Slide 6-11
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Step 1
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.
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Step 1
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T1 First and second dimension separations
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The normal gel must be stained later rounded for
each protein spot Later protein name annotation
of each stop. The protein spot must be pop-out
when user goes through the spots.
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Step 2
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 slide9-12.
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.
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Step 1
T1 Title of the step, to appear as heading of
the screen (if any)?
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Step 2
1
T2 Mass spectrometry analysis
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5
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Step 2
1
T2 Mass spectrometry analysis
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Step 2
1
T2 Mass spectrometry analysis
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Step 2
1
T2 Mass spectrometry analysis
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3
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Slide 4,5
Slide 4,5
Slide 6,11
Slide 6,11
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• Animation area
• Interaction 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.

Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1

• Question 1
• Global proteome means
• Entire protein content of the cell
• Specific proteins in the cell
• Only metabolically functional proteins
• Transcription factors
• Question 2
• E.Coli is a
• Gram positive bacteria
• Gram neutral bacteria
• Gram negative bacteria
• Mycobacteria
• Question 3
• Proteins are identified by

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APPENDIX 2
Links for further reading

• http//www.matrixscience.com/search_form_select.ht
  ml
• 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), pp931-42.
• 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), pp18-25.
• 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.

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APPENDIX 3
Summary
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.