Proteomics I

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Proteomics I

• Mass Spectrometry
• Functional Genomics by Mass Spectrometry
• (Andersen and Mann, 2000)
• FEBS Letters 480, 25-31

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Proteomics II

• Yeast Two Hybrid
• Toward a Protein-Protein Map of the Budding Yeast
• (Ito et al., 2000)
• PNAS 97(3), 1143-1147

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Mass Spectrometry

• Molecules to be analyzed, referred to as analytes
  are first ionized (usually in a vacuum),
• Newly charged molecules are introduced into an
  electric and/or magnetic field in gas phase,
• Their path through the field is a function of the
  mass to charge ratio m/z,
• m/z of the ionized species can be used to deduce
  the mass of the analyte with high precision.

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Biological Samples

• ....bringing polypeptides and nucleic acids to
  the gas phase usually degrades the molecules,

1988
matrix assisted laser desorption/ionization mass
spectrometry MALDI-MS
electrospray ionization mass spectrometry ESI-MS
2002 Nobel Prize on Chemistry, Fenn Tanaka
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Electrospray Ionization Mass SpectrometryESI-MS

• Peptides analytes, in solution, are passed
  through a charged needle that is kept at high
  electrical potential,
• the peptides are ionized,
• this disperses the the solution into a fine
  spray,
• the solvent quickly evaporates,
• peptides now in gas phase,
• Enter mass spectrometer for mass fingerprinting,
• or
• Peptide Sequencing.

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ESI-MS
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Multi-protein Complexesmud pit analysis

• ?

...i.e. nuclear pore complexes, ...i.e. cellulose
synthase complexes, ...i.e. spindle pole
apparati, ...i.e. proteins involved in the
spliceosome, etc. etc. etc.
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Post Transcriptional Modification eukaryotes

• Differential splicing can alter the final
  proteins function.

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5 mRNA Cap

• m3G Cap

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Eukaryotic Intron Excision(sequence is important)
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Spliceosomesconfer sequence specificity

• ... small nuclear RNAs (snRNAs)
• RNA molecules that act as function in
  spliceosomes,
• work in concert with gt 100 proteins to
  facilitate intron identification and removal,
• snRNPs RNA/Protein structures.

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U1 and U2snRNAs

• U1 binds to the 5 exon/intron junction.
• U2 binds to the adenosine region at the branch
  site.

Think about the required specificity for intron
identification in cells.
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Eukaryotic Intron Excision
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Base-pairing specificity of the snRNAs give the
cell control of intron excision.
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U1 snRNP(s)
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Isolate Spliceosome Complex
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Separate Ni-NTA Eluate

• ... SDS-PAGE fractionation yields 20 proteins,
• bands are in-gel digested,
• extracted,
• sent to MS-MS for ID.

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Glycerol Gradient
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b-type ions (a-amino)
y-type ions (a-carboxyl)
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MS-MS

• A. fragment at 756.9 was selected for sequencing,
• B. LEEL sequenced,
• C. AELEELEEFEFK unique match in the database.

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Whole Complex De-convolution
Prp39p (106 aa peptide)

• Prp40p (41 aa peptide)

• ...peptides can be isolated and from heterogenous
  samples, sequenced and IDd.

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Found Players

• All of the known U1-specific proteins found to
  date were identified,
• An ortholog to human U1-C was found in yeast,
  that had not been been previously identified,
• Four novel proteins were also identified.

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Establishment of Principle

• ...so, in one fell swoop, years of molecular
  genetic and biochemical research was replicated,
• and
• ...a U1-C protein, not previously detected, was
  identified as a component in the system,
• and
• ...four novel proteins have been IDd,
• not necessarily components,
• but excellent research leads.

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Use of Principle

• ...in one subsequent experiment with isolated
  human spliceosome,
• 70 spliceosome protein spots were analyzed,
• 19 novel splicing factors were identified,
• and
• ...all 19 could be cloned directly via EST
  libraries,
• and
• ...in vivo conformation of the role of these
  factors in splicing was obtained through
  co-localization studies using green fluorescence
  protein (GFP).

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Organelle(nucleolar proteins)
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Spliceosome AnalysisYeast Two-Hybrid
Proteomics II

• Nature Genetics, 1997, 16 pp 277 - 282

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GAL4 Transcription Activator
GAL4

• BD Binding Domain,
• AD Activation Domain.

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Strategy

• Bait 10 spliceosome proteins,
• Targets genomic library,
• Selections His and lacZ,
• Sequence identify ORFs,
• 2nd Round use identified preys as bait,
• Repeat.

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Results
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Wednesday

• Questions.
• Bioinformatics II

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