a highly abbreviated introduction to proteomics. a typical shotgun proteomics experiment collect...

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