2D Gel Electrophoresis

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2D Gel ElectrophoresisProtein Identification
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2D Gel Electrophoresis is one of the most
commonly used tools in proteomics
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What is Proteomics?Proteomics is the study of
the Proteome, the protein complement of the
genome
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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
• SoftwareThat Can Match MS Data With Specific
  Protein Sequences Present In Databases
• Sequence Database Providing A Catalog Of All
  Expressed Protein
• Computational Biology or Informatics

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2D gel electrophoresis

• 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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2D Gel ElectrophoresisFirst Dimension
Isoelectric FocusingSecond Dimension SDS PAGE
(apparent molecular weight)
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Sample preparation for the 1st Dimension

• Should be soluble in 1st dimension reagent
  mixture
• Should not be buffered
• Should not contain salts
• Not to exceed a certain protein content depending
  on the IEF strip used
• Sample volume

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Blank gel digest
Protein digest
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Applications
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WesternAnti-P-tyrosine
Silver stain
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p85
P85 was identified as AEBP1
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LTQ Data for Medical College

• Prepared by
• Liam Moran and Robert Shulfer
• Thermo Electron

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Ion Trap Family Tree
3D Ion Trap
2D Ion Trap
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Improvements in Performance 2D vs. 3D Ion Trap
Technology

• Spectral Ion Capacity Increased by 50-70x
• Increased ion statistics for improved spectral
  reproducibility
• Suppressed variability in signal, improved RSDs
• Ion Trapping Efficiency Increased by 11-14x
• Scan Speed greatly improved
• Ion Transmission into Ion Trap Increased by 2-5x
• Fill time for a given number of ions decreased

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Ion Max Source
Interchangeable Source Probe (ESI probe shown)
APPI probe inlet
Positional Adjusters for Source Probe
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Instrument methods (HPLC)
Flow split to 220 nl/min
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At what level can you identify a protein

• The MS and MS/MS was collected at the same time
  for all previous slides for glu-fib (data on
  LTQ.)
• Chromatographic peaks are ca. 20 sec wide.
• 324 attomoles

Full scan RIC
Full scan MSMS
Single scan
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Sample 5 (unmodified peptide)
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Sample 6 Adp-ribose modified peptide
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Sample 5 and 6
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Intact protein CRK plus
541 greater than unmodified
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Intact protein CRK plus