Toward the Characterization of Mouse Protein Complexes

Jack GreenblattUniversity of Toronto

IRC Oct. 2005

A Strategy for IDs of Stable Complexes and Weak Interactions

Tandem Affinity Purification

SDS-PAGE

Gel Bands

Trypsin Digestion

MALDI-TOFMass Spectrometry

Identification of Stably Associated Proteins

LCQ-Deca Ion TrapMass Spectrometry

Identification of Stably and Weakly Associated Proteins

ActiveProtein

ForAssays

NO GEL!

Trypsin Digestion

Purification of Tagged RNA Polymerase IIIdentification of Iwr1

no tag Rpb3

Rpb1Rpb2

Rpb3-TAP

Rpb4

Rpb5

Rpb6

Ydl115c (Iwr1)

kDa

97

66

45

31

21

Iwr1 is an evolutionarily conserved transcription elongation factor.

Proteins Identified from Rpb3-TAP by ION-TRAP Mass Spec

Iwr1

(Interacts with RNAPII)

RNA Polymerase III

RNA Polymerase I

RNA Polymerase II

Mediator

COMPASS

2005

CPF

Torpedo complex

SWR-C

Data Format for Possible A-B Interaction

purification of A-TAP

purification of B-TAP

score for identification of B by MALDI-ToF

score for identification of B by LC/MS

score for identification of A by MALDI-ToF

score for identification of A by LC/MS

TAP tags on all predicted yeast soluble proteins

TAP purifications from 4004 different strains

protein identifications from tandem MS and MALDI-ToF MS

J48 decision tree boosted stump decision treenaïve Bayes

stacking

ROC curve(MIPS protein complexes as gold standard)

ROC Curve Comparison of Yeast HTP Protein Interaction Studies

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

1- specificity (FP Rate)

Sen

siti

vity

(T

P R

ate)

Toronto DataCombined Others' DataGavinHoUetzIto

IP/MS

Y2H

Haiyuan Yu and Mark Gerstein

select mouse proteins

use phage lambda red system to tag C-terminal of mouse protein on BAC in E. coli

transform mouse ES cells with tagged BAC

identify targeting event by PCR

create tagged mice by aggregation with tetraploid embryos

affinity purify tagged mouse protein from various tissues

identify co-purifying proteins by tandem MS and MALDI-ToF MS

STRATEGY FOR MOUSE PROTEINS

attL exo bet gam cI857 (cro-attR-bioA)

PL PR Δ

Tagging E. coli Proteins in vivo Using the λred Recombination System

two affinity tags KAN

STOP CODON

Exo/Bet – λ recombinase

+

E. coli RecBCD - Exonuclease V

λ Gam

-

Temperature sensitive cI repressor – inactive at 42°C

CHROMOSOMAL ORF

_________________________________________________________________________

Protein Complexes from E. coliComparison of TAP and SPA Systems

InfC

TAP SPA

SPA TAP

Yac

L

Rpo

DS

ufD

Suf

CS

ufB

Yac

LR

poD

Suf

D

Suf

CS

ufB

RpoB,CHepA

RpoD

RpoA

YacLNusG

SufBSufD

SufC

RpsA

InfCRpsC,D

RpsG

RpsE,F,K,M,J

InfC

RNA POLYMERASE

DNA POLYMERASE

ACP

RIBOSOME ASSOCIATED

IscS

Essential

Non Essential

Interaction Network Containing the Most Highly Conserved, Soluble and Essential Proteins of E. coli

Butland et al. (2005) Nature 433: 531-537.

MR

Sem1-TAP No tag

Rpn1

Rpn2

Rpn3/Ubp6Rpt1Rpt2/Rpt3/Rpt5Rpt4/

Rpn6Rpn5/Rpn7Rpt6 Rpn8/Rpn9

Rpn10/Rpn11Rpn12

Rpn13

Sem1-TAP

kDa

97

66

31

21

14

B. DSS1-SPANo tagMR

kDa

97

66

45

31

21

S1 (Rpn2)S2 (Rpn1)

S3 (Rpn3)S4 (Rpt2)S5A (Rpn10)S6B (Rpt3)

S7 (Rpt1) S8 (Rpt6) S9 (Rpn6)S10A (Rpn7)S10B (Rpt4)S11 (Rpn9)

The BRCA2 Interactor, DSS1, and its Yeast Homolog, Sem1, are Proteasome Subunits

Benoit Coulombe, 2005

Tags Tested for Toxicity in Mouse ES Cells and Purification Efficiency

Tag Purification Efficiency Toxic in ES cells?

TAP (CBP + pA) + yes

SPA (CBP + 3 X FLAG) + yes

FA (3 X FLAG + pA) + somewhat

HF (6 X HIS + 3 X FLAG) ++ no

SF (SBP + 3 X FLAG) ++ nd

TA

P SP

A

FA HF SF Mr Mr

SP

A

FA HF FH SF

tagged GFP

tagged GFP

94

68

43

30

21

Purification from E. coli of GFP with Various Tandem Affinity Purification Tags

FA HF#1 HF#6Mr HF#6

liver

mu

scle

brai

n

kidn

ey

hea

rt lun

g testis

embryos

HF#1

6His-tagged GFP

TEV protease

3FLAG-tagged GFP

94

68

43

30

21

ES cells

Purification of Tagged GFP from Mouse ES Cells, Embryos, and Various Tissues

HF PneoLhom Rhom

Primers for wild-type allele

Primers for homologous recombination

FRT site for excision by FLPe

Coding sequence of transcript

ATG

Organization of Cassette Used to Tag Mouse Proteins

tag

KEY PEOPLE

Greenblatt LabJoyce Li

Nevan Krogan

Andrew Emili Gareth Butland

Janet RossantMarina Gertsenstein