1. A highly abbreviated introduction to proteomics

2. A typical shotgun proteomics experiment
Collect tens of thousands of MS/MS spectra
Can identify >1,000 proteins from cell lysate

3. Orbi video:

4. Shotgun proteomics identifies proteins from the fragmentation mass spectra of their constituent peptides
b & y
ions
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:

5. 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:

6. Mass spectrometry strategies for measuring absolute protein abundances
for 100’s to 1000’s of proteins
adapted from Vogel & Marcotte
Nature Biotechnology , 825-6

7. & the current state-of-the-art …
Each K peptides, from ~10,000 proteins spanning ~7 orders of magnitude in abundance

8. A highly abbreviated introduction to
large-scale protein interaction screens

10. X-ray structure
of ATP synthase
Schematic
version
Network
representation a b g d b2 e a
c12
Total set = protein complex
Sum of direct + indirect
interactions

12. High-throughput yeast two-hybrid+
DBD
Bait
Prey
Act
DNA binding
domain
Transcription
activation
domain
Prey
Act
Core transcription
machinery
Bait
DBD
transcription
operator or
upstream activating
sequence
Reporter gene

13. High-throughput yeast two-hybrid
Haploid yeast
cells expressing
activation domain-
prey fusion proteins
Diploid yeast
probed with
DNA-binding domain-
Pcf11 bait
fusion protein

14. High-throughput complex mapping by mass spectrometry
Tag
Bait
Affinity
column
protein 1
protein 2
SDS-
page
protein 3
Trypsin digest,
identify peptides by
mass spectrometry
protein 4
protein 5
protein 6

15. 493 bait proteins
3617 “interactions”

16. A variant: tandem affinity purification (TAP)
Tag1
Tag2
Bait
Affinity
column2
protein 1
Affinity
column1
protein 2
SDS-
page
protein 3
protein 4
+ protease
protein 5
protein 6
Trypsin digest,
identify peptides by
mass spectrometry
Affinity
column1

18. Estimating accuracy with a well-determined reference set of interactions

19. Where we were, more or less, until recently in terms of PPI maps

20. 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

21. >2,000 biochemical fractions,
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)

22. The profiles cover > ½ the experimentally verified proteome
& proteins within the same stable complexes co-elute
Havugimana,
Hart, et al.,
Cell (2012)

23. Turning separations into complexes
1) One separation, #13 of many
Cluster
4) Inferred complexes
~5600 proteins
~120
fractions
... 59 60 61 62 63 64
Co-separation of the exocyst complex
exoc1
exoc2
exoc3
exoc4
exoc5
exoc6
exoc7
exoc8
3) Inferred interactions
high correlation >> more likely in complex
2) Pairwise protein correlations
Machine learning
(SVM, Ensemble methods)
Hurdle is false positives: since you’re searching the entire space of possible shared complex memberships, where true co-memberships are extremely sparse, high scorers are dominated by false positives. Must use external data to
2b) External data
Co-expression, shared protein domains, much more (HumanNet)
Other AP-MS datasets (Guruharsha 2011, Malovannaya 2011)

24. Guiding and testing the reconstruction with known complexes
Havugimana,
Hart, et al.,
Cell (2012)

25. 13,998 high-confidence physical interactions / 3,011 proteins
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)

26. 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)

27. The human protein complexes are also strongly enriched
for genes linked to the same diseases and phenotypes
Havugimana,
Hart, et al.,
Cell (2012)

28. 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
Online Journal 7(2): 8

29. Our current state of the art animal complex map
Cuihong Wan
Blake Borgeson
w/ Andrew Emili’s lab

30. Our current state of the art animal complex map
Extending the map
Now 7 animals, >65 separations, nearly 7,000 mass spec experiments
>3,500 fractions
~12,000
proteins
Our current state of the art animal complex map
>3,000
fractions
~9,000
proteins
Cuihong Wan
Blake Borgeson
w/ Andrew Emili’s lab