Lab 6.4: Computational Tools for Protein Identification

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Lab 6.4 Computational Tools for Protein
Identification

• Dr. Joanne Fox
• Day 6 Saturday February 21st, 2004
• 1545pm 1715pm

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Lab 6.4 Objectives

• As a result of the diversity of features encoded
  by protein sequences there have been a plethora
  of tools developed to identify and predict
  protein features. In this lab, you will get an
  opportunity to choose the appropriate
  computational tools to aid you in identifying and
  describing proteins isolated from a 2D-gel. You
  will get hands-on experience using tools for
  protein identification from a peptide mass
  fingerprint.

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2D Gel Data
Spot 1
Mr 210 185 135 80 30 15

• Spot Features
• Spot 1
• Mw210,000
• pI5.3
• Spot 2
• Mw9,000
• pI4.7

Spot 2
4.0 5.0 6.0 7.0 8.0
pI
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Lab 5.2 Worked Example 1

• Strategy for Identification of Spot 1
• Use pI and Mw to generate list of candidate
  proteins

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http//ca.expasy.org/
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With your group, you can repeat this analyses and
try and identify a candidate protein list for
spot 2.Worked Example 1 Task 2.
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Lab 6.4 Worked Example 2

• Peptide mass fingerprinting (PMF) lists were
  generated from mass spectrometric analysis of a
  trypsin-digested, liquid chromatography (LC)
  separated fractions
• Data from cellular extracts of E. coli and S.
  cerevisiae

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Using Computational Tools to Predict Protein
Identity with the PMF data

• Compare and contrast these two different tools
  for protein identification
• Mascot
• http//www.matrixscience.com/
• PeptIdent
• http//us.expasy.org/tools/peptident.html

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Lets start with Mascot

• Mascot
• Go to http//www.matrixscience.com/
• On the Matrix Science home page, click on the
  Mascot link
• On the Mascot search page, click on the Peptide
  Mass Fingerprint link
• PLEASE WORK IN PAIRS Be a good citizen in the
  online bioinformatics world NOT GOOD ETIQUETTE
  TO SEND 50 EQUIVALENT JOBS TO WEB SERVER

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Click on the blue Query and you find out that
the PMF data must be one per line. You can get
this data off the Day 6 link on the course
website cut and paste it here
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Each team should now try use Mascot and PeptIdent
with the PMF data found on the course website
(see Day 6 link).Try and complete Tasks 2 and
3 of Worked Example 2 in Lab 6.4!
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Some Advice

• Be methodical in your searches.
• Compare your data within your group, go at your
  own pace, we will have time to review and
  wrap-up.
• The goal here is not to get the exact same
  protein identifications from each of the PMF
  tools but to illustrate how significantly the
  parameters and the different programs can affect
  the outcome.
• Check the Lab 6.4 handout for advice on which
  parameters to change from defaults.