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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From the abstract This is the first time that
the components of a multi-protein complex from an
organism with sequenced genome have been
characterized by mass spectrometry. The
technique should be applicable to any protein
complex that can be biochemically purified from
an organism whose genome is known.
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Post Transcriptional Modification eukaryotes

• Splicing produces final translation template
  and differential splicing may 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 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)
http//www.nature.com/nature/journal/v458/n7237/fu
ll/nature07851.htmlB2
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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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(No Transcript)
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Monday

• Group 1,
• Gut Metagenomics.