Transcript PPIIntro-4-2013x

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/
Shotgun proteomics identifies proteins from the fragmentation mass
spectra of their constituent peptides
Peptide
fragmentation
Actual peptide
tandem (MS/MS)
mass spectrum
Idealized peptide
tandem (MS/MS)
mass spectrum
from database
Idealized peptide
tandem (MS/MS)
mass spectrum
with PTM
(phosphoserine)
Marcotte (2007) Nature Biotechnology 25:755-757
b&y
ions
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 spiking
in isotopically shifted peptide
standards for proteins of interest
Mallick & Kuster (2010) Nature Biotechnology 28:695-709
Mass spectrometry strategies for measuring absolute protein abundances
for 100’s to 1000’s of proteins
adapted from Vogel & Marcotte
Nature Biotechnology 2009 27, 825-6
& the current state-of-the-art …
Each 100-200K peptides, from ~10,000 proteins spanning ~7 orders of magnitude in abundance
A highly abbreviated introduction to
large-scale protein interaction screens
X-ray structure
of ATP synthase
Schematic
version
Network
representation
a
b
d
g
b2
e
a
Total set = protein complex
Sum of direct + indirect
interactions
c12
High-throughput yeast two-hybrid
DBD
Bait
DNA binding
domain
+
Prey
Act
Transcription
activation
domain
Core transcription
machinery
transcription
operator or
upstream activating
sequence
Reporter gene
High-throughput yeast two-hybrid
Haploid yeast
cells expressing
activation domainprey fusion proteins
Diploid yeast
probed with
DNA-binding domainPcf11 bait
fusion protein
High-throughput complex mapping by mass spectrometry
Tag
Bait
Affinity
column
SDSpage
protein 1
protein 2
protein 3 Trypsin digest,
protein 4 identify peptides by
protein 5 mass spectrometry
protein 6
493 bait proteins
3617 “interactions”
A variant: tandem affinity purification (TAP)
Tag1 Tag2
Bait
Affinity
column2
Affinity
column1
SDSpage
+ protease
protein 1
protein 2
protein 3
protein 4
protein 5
protein 6
Trypsin digest,
identify peptides by
mass spectrometry
Affinity
column1
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
~3,500 affinity purification
experiments
~11K interactions /
~2.3K proteins
 spans 556 complexes
Still daunting for the
human proteome
Guruharsha et al. (2011) Cell 147, 690–703
Finding stable protein assemblies by native separations and quantitative mass spec.
>2,000
biochemical
fractions,
including
replicates
>9,000 hours
mass spec
machine
time
Havugimana,
Hart, et al.,
Cell (2012)
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
1) One separation, #13 of many
4) Inferred complexes
~5600 proteins
...
~120
fractions
59
60
61
62
63
64
exoc1
exoc2
exoc3
exoc4
exoc5
exoc6
exoc7
exoc8
Co-separation
of the exocyst
complex
2) Pairwise protein correlations
2b) External data
•Co-expression, shared protein
domains, much more (HumanNet)
•Other AP-MS datasets (Guruharsha
2011, Malovannaya 2011)
Machine learning
(SVM, Ensemble
methods)
high correlation
>> more likely in complex
Cluster
3) Inferred interactions
Guiding and testing the reconstruction with
known complexes
Havugimana,
Hart, et al.,
Cell (2012)
A reference map of human protein complexes
13,998 high-confidence physical interactions / 3,011 proteins
Defines >600 complexes: >100 heterodimers, >500 with ≥3 components
Havugimana,
Hart, et al.,
Cell (2012)
In yeast, phenotypes reflect biological modules.
e.g., lethality is tied not to the protein,
but to the molecular machine
small
nucleolar
ribonucleoprotein
complex
SAGA
transcription factor/
chromatin remodeling
complex
TAFIID
complex
protein
phosphatase
2A complex
Essential gene
Nonessential gene
Hart, Lee, & Marcotte, BMC Bioinformatics 8:236 (2007)
The human protein complexes are also strongly enriched
for genes linked to the same diseases and phenotypes
Havugimana,
Hart, et al.,
Cell (2012)
The complexes are strongly enriched for genes linked to the
same diseases, e.g., as for Cornelia de Lange Syndrome
prweb.com
Now confirmed by Deardorff et al., Am. J. Hum. Genet. 90, 1014–1027
Dermatology
Our current state of the art animal complex map
Cuihong Wan
Blake Borgeson
w/ Andrew Emili’s lab
Our current state
Extending
of the art
theanimal
map complex map
Now 7 animals, >65 separations, nearly 7,000 mass spec experiments
>3,000
fractions
>3,500 fractions
~9,000
proteins
~12,000
proteins
Cuihong Wan
Blake Borgeson
w/ Andrew Emili’s lab