Protein Analysis Techniques

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Protein AnalysisTechniques
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Name SomeAnalysis Techniques

• Polyacrylamide gel electrophoresis
• 2-D PAGE
• HPLC
• Mass Spectrometry

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How Is ProteomeDifferent From Genome?
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Technology IsDifferent

• Technologies to separate large numbers of
  proteins, identify them and study their structure
  and modifications are not straightforward.
• DNA Applied Biosystem high-throughput DNA
  sequencer

PE 3700 Sequencer
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Technology IsDifferent

• Protein- Picks and Shovels
• Two Dimensional Electrophoresis
• 25 years old
• Only separation technology which provides highly
  purified proteins separated in a simple parallel
  process is 2-D electrophoresis

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What LabTechnologies Will Drive The Proteomics
Revolution?

• Two-dimensional gel electrophoresis?
• Capillary Electrophoresis?
• Protein chips?
• Mass spectrometer?
• Something yet to be invented?

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Mass Spectrometry

• What is it?
• Why dont we have one?
• What is it used for?
• Inputs and outputs
• Where to find more info

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What Is It?

• Analytical technique
• Weighs any particular molecule or component
  molecules of a mixture
• Peptide fingerprinting

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Two Main Parts

• Ion Source
• Introduces sample into machine
• Apparatus to measure mass of introduced ions

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Simulation

• http//www.colby.edu/chemistry/OChem/DEMOS/MassSpe
  c.html

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Two Main Types

• Matrix assisted laser desorption/ionization time
  of flight mass spectrometry or
• MALDI-TOF MS
• Generates ions from solid phase samples
• Flight tube for measuring mass

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Two Main Types

• Electrospray ionization mass spectrometry
  (ESI-MS)
• Generates ions from liquid samples

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Dramatic Improvements

• Ion reflector and delayed extraction
• Allows isotopic resolution of peptides and high
  mass accuracies

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What Is It UsedFor?

• Peptide Mass Fingerprinting
• Run a 2 D PAGE gel, transfer to nitrocellulose
• (or PVDF)
• Remove an area corresponding to one protein
• Treat target protein with protease (trypsin,
  pepsin, etc)
• Perform mass spectrometry
• You have a peptide mass fingerprint

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• MEQRITLKDYAMRFGQTKTAKDLGVYQSAINKAIHAGRKIFLTINADGSV
  YAEEVKPFPSNKKTA

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• After proteolytic digestion
• TA
• MEQR
• DYAMR
• FGQTK
• DLGVYQSAINK
• IFLTINADGSVYAEEVKPFPSNK

• TAK
• ITLK
• FGQTK
• AIHAGR

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• Compare the fingerprint to a protein fingerprint
  database to determine if it is a known protein
  
• Example
• Of 150 proteins analyzed from 2-D separations of
  S. cerevisiae, 80 were identified without
  ambiguity by MS mass fingerprinting
  

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• What you know about a subject is not nearly as
  significant as knowing where to find out what you
  need to know about that subject.

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Protein Function In The Post-Genomic Era
DAVID EISENBERG, et al. Nature 405, 823 - 826
(2000)