a highly abbreviated introduction to proteomics
DESCRIPTION
A highly abbreviated introduction to proteomics. A typical shotgun proteomics experiment. Collect tens of thousands of MS/MS spectra Can identify >1,000 proteins from cell lysate. Orbi video: http://apps.thermoscientific.com/media/SID/LSMS/Video/webinar/orbitrap_elite/animation/. - PowerPoint PPT PresentationTRANSCRIPT
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A highly abbreviated introduction to proteomics
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A typical shotgun proteomics experiment
Collect tens of thousands of MS/MS spectraCan identify >1,000 proteins from cell lysate
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Orbi video:http://apps.thermoscientific.com/media/SID/LSMS/Video/webinar/orbitrap_elite/animation/
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Shotgun proteomics identifies proteins from the fragmentation mass spectra of their constituent peptides
Marcotte (2007) Nature Biotechnology 25:755-757
Peptidefragmentation
b & yions
Actual peptidetandem (MS/MS)mass spectrum
Idealized peptidetandem (MS/MS)mass spectrumfrom database
Idealized peptidetandem (MS/MS)mass spectrumwith PTM(phosphoserine)
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One common strategy for relative quantification =using isotopically labeled samples(e.g. 15N vs. 14N, 13C vs. 12C, etc.)
SILAC = stable isotope labeling with amino acids in cell culture
iCAT = isotope tags on cysteines
iTRAQ = isobaric labels on cysteines(same mass, different isotopes)
AQUA = absolute quantification by spikingin isotopically shifted peptidestandards for proteins of interest
Mallick & Kuster (2010) Nature Biotechnology 28:695-709
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Mass spectrometry strategies for measuring absolute protein abundancesfor 100’s to 1000’s of proteins
adapted from Vogel & MarcotteNature Biotechnology 2009 27, 825-6
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Each 100-200K peptides, from ~10,000 proteins spanning ~7 orders of magnitude in abundance
& the current state-of-the-art …
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A highly abbreviated introduction tolarge-scale protein interaction screens
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a b
e
gd
b2
c12a
Networkrepresentation
X-ray structureof ATP synthase
Schematicversion
Total set = protein complexSum of direct + indirect
interactions
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Bait
DBD
High-throughput yeast two-hybrid
Bait Prey
DNA bindingdomain
Transcriptionactivationdomain
+
operator orupstream activating
sequence
Reporter gene
transcription
Core transcriptionmachinery
DBD Act
Prey
Act
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Diploid yeastprobed with
DNA-binding domain-Pcf11 bait
fusion protein
Haploid yeastcells expressing
activation domain-prey fusion proteins
High-throughput yeast two-hybrid
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protein 1protein 2protein 3protein 4protein 5protein 6
BaitTag
Affinitycolumn
SDS-page
Trypsin digest, identify peptides bymass spectrometry
High-throughput complex mapping by mass spectrometry
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493 bait proteins
3617 “interactions”
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BaitTag2
Affinitycolumn1
A variant: tandem affinity purification (TAP)
Tag1
Affinitycolumn1
+ protease
Affinitycolumn2
protein 1protein 2protein 3protein 4protein 5protein 6
SDS-page
Trypsin digest, identify peptides bymass spectrometry
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Estimating accuracy with a well-determined reference set of interactions
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Where we were, more or less, until recently in terms of PPI maps
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The current state-of-the-art in animal PPI maps
Guruharsha et al. (2011) Cell 147, 690–703
~3,500 affinity purification experiments
~11K interactions / ~2.3K proteins
spans 556 complexes
Still daunting for thehuman proteome
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>2,000 biochemical
fractions,including replicates
>9,000 hoursmass spec machine
time
Havugimana,Hart, et al.,Cell (2012)
Finding stable protein assemblies by native separations and quantitative mass spec.
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The profiles cover > ½ the experimentally verified proteome & proteins within the same stable complexes co-elute
Havugimana,Hart, et al.,Cell (2012)
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Turning separations into complexes
~5600 proteins
1) One separation, #13 of many
~120fractions
exoc
1ex
oc2
exoc
3ex
oc4
exoc
5ex
oc6
exoc
7ex
oc8
Co-separation of the exocyst complex
596061626364
...
Cluster
4) Inferred complexes
3) Inferred interactions
high correlation >> more likely in complex
2) Pairwise protein correlationsMachine learning(SVM, Ensemble methods)
2b) External data• Co-expression, shared protein
domains, much more (HumanNet)• Other AP-MS datasets
(Guruharsha 2011, Malovannaya 2011)
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Guiding and testing the reconstruction with known complexes
Havugimana,Hart, et al.,Cell (2012)
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13,998 high-confidence physical interactions / 3,011 proteinsDefines >600 complexes: >100 heterodimers, >500 with ≥3 components
A reference map of human protein complexes
Havugimana,Hart, et al.,Cell (2012)
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SAGAtranscription factor/
chromatin remodelingcomplex
TAFIIDcomplex
Nonessential geneEssential gene
proteinphosphatase2A complex
smallnucleolar
ribonucleoproteincomplex
In yeast, phenotypes reflect biological modules.e.g., lethality is tied not to the protein,
but to the molecular machine
Hart, Lee, & Marcotte, BMC Bioinformatics 8:236 (2007)
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The human protein complexes are also strongly enrichedfor genes linked to the same diseases and phenotypes
Havugimana,Hart, et al.,Cell (2012)
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prweb.com
DermatologyOnline Journal 7(2): 8Now confirmed by Deardorff et al., Am. J. Hum. Genet. 90, 1014–1027
The complexes are strongly enriched for genes linked to the same diseases, e.g., as for Cornelia de Lange Syndrome
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Cuihong WanBlake Borgeson
w/ Andrew Emili’s lab
Our current state of the art animal complex map
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Extending the mapNow 7 animals, >65 separations, nearly 7,000 mass spec experiments
>3,500 fractions
~12,000proteins
Our current state of the art animal complex mapNow 7 animals, >65 separations, nearly 7,000 mass spec experiments
~9,000proteins
>3,000fractions
Cuihong WanBlake Borgeson
w/ Andrew Emili’s lab
Our current state of the art animal complex map