Protein Identification And Amino Acid Sequencing

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Protein Identification AndAmino Acid Sequencing

• Protein Identification Using MALDI-TOF Mass
  Spectrometry Proteomics
• Use of Traditional Edman Chemistry for Amino Acid
  Sequencing
• Use of Modern Time Mass Spectrometry for amino
  Acid Sequencing

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• Tools of Proteomics
• Analytical Protein Separation Technologies
• ex 2D-SDS-PAGE, HPLC, CE, multidimensional
  techniques
• 2. Mass Spectrometry
• ex MALDI-TOF, ESI-MS-MS, Iontrap, Triplequad
• 3. SoftwareThat Can Match MS Data With Specific
  Protein Sequences Present in Databases
• 4. Complete Genomic Sequence Database providing A
  Catalog Of All Expressed Proteins

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• 2D-Gel ElectrophoresisA Key Technology Driving
  Proteomics
• Reproducible 2D Gel electrophoresis
• Staining and Scanning of the Gels
• Proteolytic digestion of the protein(s) within
  selected spot(s)
• Mass Spectrometry for Identification
• Databases Searching

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Pros of MALDI-TOF MS

• Soft ionization-molecules stay intact
• Broad mass range- 400 Da 400kDa
• Mixtures are OK
• High sensitivity
• Data is easy to interpret
• Fast
• Easy to use and maintain

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Cons of MALDI-TOF MS

• Limited to measuring peptide masses. MALDI-TOF
  instruments are not suited for peptides ion
  fragmentation which provides amino acid sequence
  data.
• Success of analysis is highly dependent on
  quality of sample. Contaminants such as
  detergent, buffer, DTT, urea, etc inhibit
  peptide ionization.

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Amino Acid sequencing
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Sample Preparation

• General Strategy (Protein Purification)
• Choice of Cleavage (Chemical, Enzymatic)
• Peptide Purification
• Checking for Purity
• Quantitation

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Edman Degradation

• Amounts needed
• Chemistry
• Data analysis

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Peptide sequencing by LC/mass spectrometry
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In contrast to MALDI where peptides are
introduced in a dry form (crystalline admixture
of peptide sample and matrix) into the analyzer,
peptides to be analyzed by ESI are in aqueous
solution
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• A1 A2 A3 A4
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•  b-ions..y-ions

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2D vs. 3D

Ion Transfer
Tube
Skimmer

Tube Lens
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