Chem 415 Amino Acids and Peptides

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Chem 415 Amino Acids and Peptides
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How to Analyze a protein

• Figure out its size
• Equilibrium sedimentation
• Gel exclusion chromatography
• SDS PAGE electrophoreses
• reducing and non reducing conditions

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How to Analyze a protein

• Sequence
• AA composition \Acid Hydrolysis
• Reverse phase chromatography
• Sequencing
• Strategies
• DNA
• Protein

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N Terminal analysis
FDNB Yields DNP peptide bright yellow
Dansyl Chloride Yields dansylated
peptide Fluorescent
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C-Term analysis

• Current Technology is poor
• Hydrazinolysyis
• results in multiple hydrozides that are difficult
  to sort out
• Carboxypetidase
• sequentially releases AA from carboxy end
• AA Dependent

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Stratagy

• Break into pieces
• Sequence pieces
• Break into different set of pieces
• Sequence pieces
• look for overlap between the two sets of pieces
• repeat if necessary

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A bit of history

• Sanger (two Nobel prizes
• Published Insulin Sequence in 1955
• partial hydrolysis technique that left him with
  di Tri tetra peptides. Painstakingly seperated
  and then n term labeled so that he could
  distinguish the two aa in the peptide

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A bit of history

• Sanger (two Nobel prizes
• Determine the sequence a peptide if it yields the
  following di-peptides
• TY PT DE PH AS YP EP SD
• dansylation of the whole peptide followed by
  complete hydrolysis yielded dansyl A

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Edman degradation

• Described in 1950 using wet chemistry
• 3-5 cycles
• Should have won a Nobel prize major advancement
  in sequencing
• Current spinning cup machines sequence 30 -40 AA
  at a one go. (equals 90-100 bp)

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Tools to sequence large proteins
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Mass Spec Methods

• May be use full on mixtures separated by 2 D
  electrophoresis
• works by fragmenting the peptide at the peptide
  bond which is the most fraglile of the bonds
• each AA Gives a Characteristic mass change of the
  peptide

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MALDI TOF
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MALDI TOF
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MALDI TOF
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Mass Spec Sequencing
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Disulfide location

• Diagonal maps
• Reverse phase Chromotography

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Disulfide location2 D electrophoresis
Origin
Initial electrophoresis
Electrophoresis second direction
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Disulfide location2 D electrophoresis
Origin
Initial electrophoresis
Electrophoresis after Oxidation