Proteomics I

Mass Spectrometry

Functional Genomics by Mass Spectrometry(Andersen and Mann, 2000)

FEBS Letters 480, 25-31

optional

Proteomics II

Yeast Two Hybrid

Toward a Protein-Protein Map of the Budding Yeast(Ito et al., 2000)

PNAS 97(3), 1143-1147

optional

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.

Biological Samples

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

matrix assisted laser desorption/ionization mass spectrometryMALDI-MS

1988

electrospray ionization mass spectrometryESI-MS

2002 Nobel Prize on Chemistry, Fenn & Tanaka

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.

ESI-MS

Multi-protein Complexes“mud 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.

Post Transcriptional Modification eukaryotes

Differential splicing can alter the final proteins function.

5’ mRNA Cap

m3G Cap

Eukaryotic Intron Excision(sequence is important)

Spliceosomesconfer sequence specificity

... small nuclear RNAs (snRNAs):

– RNA molecules that act as function in spliceosomes,

…work in concert with > 100 proteins to facilitate intron identification and removal,

– snRNPs: RNA/Protein structures.

U1 and U2snRNAs

U1 binds to the 5’ exon/intron junction.

U2 binds to the adenosine region at the branch site.

Psuedouradine (

Think about the required specificity for intron identification in cells.

Eukaryotic Intron Excision

Base-pairing specificity of the snRNAs give the cell control of intron

excision.

U2 and U6, snRNAsproteins not shown

U1 snRNP(s)

Isolate Spliceosome Complex

Anti-m3G Antibody Chromotography

...RNA processing includes the capping of the 5’ end by 7-methylguanosine triphosphate,

…antibodies are available for this molecule,

…all molecules with a capped RNA can be co-precititated.

Histidine Tag Snp1Nickel Nitrilotriacetic Acid (Ni-NTA) Affinity Chromotography

...Snp1 has been identified as a U1 specific snRNP,

...the functional Snp1, in the experiment (yeast), is carried on a plasmid,

...it was modified by a hexahistidine addition to the N-terminus,

...the exposed his-tag binds to nickel on a chromatography column, is washed off with imidazole.

Separate Ni-NTA Eluate

... SDS-PAGE fractionation yields 20 proteins,

• bands are in-gel digested,• extracted,• sent to MS-MS for ID.

Glycerol Gradient

b-type ions (-amino) y”-type ions (-carboxyl)

MS-MS

A. fragment at 756.9 was selected for sequencing,

B. LEEL sequenced,

C. AELEELEEFEFK unique match in the database.

Other partially sequenced peptides correspond

(underlined).

Whole Complex De-convolution

...peptides can be isolated and from heterogenous samples, sequenced and ID’d.

* Prp39p (106 aa peptide)

• Prp40p (41 aa peptide)

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.

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 ID’d,– not necessarily components,

– but excellent research leads.

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).

Organelle(nucleolar proteins)

Spliceosome AnalysisYeast Two-Hybrid

Nature Genetics, 1997, 16 pp 277 - 282

Proteomics II

GAL4 Transcription Activator

BD: Binding Domain,

AD: Activation Domain.

GAL4

Strategy

Bait: 10 spliceosome proteins,

Targets: genomic library,

Selections: His+ and lacZ+,

Sequence: identify ORFs,

2nd Round: use identified preys as bait,

Repeat.

Results

Wednesday

• Questions.

• Bioinformatics II