A highly abbreviated introduction to proteomics. A typical shotgun proteomics experiment Collect...
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A highly abbreviated introduction to proteomics
A typical shotgun proteomics experiment
Collect tens of thousands of MS/MS spectraCan identify >1,000 proteins from cell lysate
Orbi video:http://apps.thermoscientific.com/media/SID/LSMS/Video/webinar/orbitrap_elite/animation/
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)
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
Mass spectrometry strategies for measuring absolute protein abundancesfor 100’s to 1000’s of proteins
adapted from Vogel & MarcotteNature Biotechnology 2009 27, 825-6
Each 100-200K peptides, from ~10,000 proteins spanning ~7 orders of magnitude in abundance
& the current state-of-the-art …
A highly abbreviated introduction tolarge-scale protein interaction screens
a b
e
gd
b2
c12a
Networkrepresentation
X-ray structureof ATP synthase
Schematicversion
Total set = protein complexSum of direct + indirect
interactions
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
Diploid yeastprobed with
DNA-binding domain-Pcf11 bait
fusion protein
Haploid yeastcells expressing
activation domain-prey fusion proteins
High-throughput yeast two-hybrid
protein 1protein 2protein 3
protein 4protein 5protein 6
BaitTag
Affinitycolumn
SDS-page
Trypsin digest, identify peptides bymass spectrometry
High-throughput complex mapping by mass spectrometry
493 bait proteins
3617 “interactions”
Bait
Tag2
Affinitycolumn1
A variant: tandem affinity purification (TAP)
Tag1
Affinitycolumn1
+ protease
Affinitycolumn2
protein 1protein 2protein 3
protein 4protein 5protein 6
SDS-page
Trypsin digest, identify peptides bymass spectrometry
Estimating accuracy with a well-determined reference set of interactions
Where we were, more or less, until recently in terms of PPI maps
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
>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.
The profiles cover > ½ the experimentally verified proteome & proteins within the same stable complexes co-elute
Havugimana,Hart, et al.,Cell (2012)
Turning separations into complexes
~5600 proteins
1) One separation, #13 of many
~120fractions
exoc
1
exoc
2
exoc
3
exoc
4
exoc
5
exoc
6
exoc
7
exoc
8
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)
Guiding and testing the reconstruction with known complexes
Havugimana,Hart, et al.,Cell (2012)
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)
SAGAtranscription factor/
chromatin remodelingcomplex
TAFIIDcomplex
Nonessential gene
Essential 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)
The human protein complexes are also strongly enrichedfor genes linked to the same diseases and phenotypes
Havugimana,Hart, et al.,Cell (2012)
prweb.com
DermatologyOnline Journal 7(2): 8
Now 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
Cuihong WanBlake Borgeson
w/ Andrew Emili’s lab
Our current state of the art animal complex map
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