Introduction to Proteomics

1
Proteomics
Session 12 Other applications by 2-DE and Mass
2
Application by 2-DE and Mass

• 1. Determination of amino acid sequence
• 2. Differential display proteomics (comparison
  proteomics)
• 3. Posttranslational modification

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1. Determination of amino acid sequence
4
Using 1-DE and LC/MS/MS for amino acid sequence
determination
5
Basic elements for MS and tandem MS (Review again)
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2. Differential display proteomics (comparison
proteomics)
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Differential display proteomics

• Difference between global profiling proteomics
  and differential display proteomics
• Protein expression profiling global profiling
  proteomics differential display proteomics

8
General approach
Visualized by 2-DE gel staining
A
Excise spot elute digest Extract peptides MS
analyze Protein identification
9
Isotopic labeling another strategy for
differential display proteomics
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Isotopic labeling approach

• In vivo labeling
• Microorganisms 15N or 13C
• Mammalian cell lines amino acid with isotope
  deuterium
• In vitro labeling (chemical or enzymatic)
• Water exchange (18H2O)
• Esterification (1H3 or 2H3 methanol)
• Isotopically coded affinity tags (ICAT)
• DIGE system

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In vivo labeling
mix
Cells A
extraction
reduction
alkylation
digestion
Cells B
separation
MS IDENTIFICATION and QUANTITATION
Enriched Medium (13C, 15N or amino acid with
deuterium)
Oda Y, et al. P Natl. Acad. Sci. USA 96
6591-6596 (1999)
Pasa-Tolic L, et al. J Am Chem. Soc. 121
7949-7950 (1999)
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In vitro labeling (proteolytic labeling)
18O water
Cells A
alkylation
extraction
reduction
digestion
Cells B
separation
MS IDENTIFICATION and QUANTITATION
Stewart T. et al. R.ap. Comm.. Mass Spectrom.
15(24) 2456. (2001)
Yao XD et al. Anal. Chem.73(13)2836-2842. 2001
13
How 18O incorporated
R-C NH-R
R-C NH-R
H2 (18) O
enzyme
H2O
enzyme
R-C (18) OH
R-C OH

H2N-R

H2N-R
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In vitro labeling ISOTOPE-CODE AFFINITY TAG (ICAT)
Linker containing the Isotope Tag
Reactive Group
Biotin Tag
Gigy et al.Nat. Biotech. 17 (10) 994-999 (1999)
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The ICAT strategy
LC-MS
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Spec. for ICAT regeant

• They attach a label to peptides that contain only
  a specific type of residue, (e.g. cysteine).
• They allow selective affinity purification of the
  labeled peptides, (e.g. through biotin).
• They enable quantitation through differential
  isotope coding.
• They provide pairs of fragment ions for precursor
  ion searching.

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Problems with ICAT

1. Expensive ( 500/sample)
2. Selectivity for cysteine may be a problem (8
   peptides contain Cys)
3. Isotopic effect in high resolution
   chromatography. Zhang RJ et al. Anal. Chem. 73
   5142-5149 (2001)
4. Quantitation accuracy (?) .../- 10

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DIFFERENTIAL GEL ELECTROPHORESIS (DIGE)
Image A
Cy3
Cells A
lE-Cy3
Gel analysis (overlay and
quantitation)
mix
Gel imaging
2-D PAGE
lE-Cy5
Cells B
Image B
Cy5
MS IDENTIFICATION
Unlu et al. Electrophoresis 18 2071-77. (1997)
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DIGE for detection of cancer marker
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Continued..
lE-Cy3
lE-Cy5
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3. Posttranslational modification
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Post-translational modification (PTM)

• PTM is more difficult than peptide sequencing
  because
• dynamic range is low
• Isolation of PTM peptides is required
• PTM is frequent labile
• PTM is frequent transient in nature
• Two major PTM
• Phosphorylation
• Glycosylation

23
Comparison table of three methods
forphosphopeptide analysis
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Summary of phosphoprotein/phosphopeptide
enrichment
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Phosphopeptide enrichment

• Ab affinity chromatography (only Anti p-Tyr ab
  available)
• IMAC (immobilized metal affinity chromatography)
• Utilization of the carbodiimide condensation.
  (ethanolamine)
• Utilization of beta-elimination reaction of
  p-Ser, p-Thr

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carbodiimide condensation of phosphopeptide
TRENDS in Biotechnology Vol 20 No.t June 2002
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Alternate strategy for isolation phosphopeptides I
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Beta elimination of phosphopeptide
TRENDS in Biotechnology Vol 20 No.t June 2002
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Alternate strategy for isolation phosphopeptides
II