High Throughput Protein, Protein-Protein Interaction, and Metabolite Assays CE

Claire Ouimet, Jing Nie, Erik Guetschow,

Shuwen Sun & Shi Jin

Collaborators: David Lombard, Jason Gestwicki

High-speed Electrophoresis

Jacobson. Anal Chem 1998, 70, 3476 Moore. Anal Chem 1993, 65, 3550

Tao. Anal Chem 1998, 70, 4015

-High E

-Short Distance

-Control Joule Heat (dimensions,

conductivity)

-Narrow injection

-Sensitive detection

Applications of Fast Electrophoresis

• Western blot

• High Throughput

Screening

• Non-covalent

interactions

• “Sensing”

• 2-D Separations

8 10 12 14 16 180

1

2

3

4

Time (s)

*

* UNC Ramsey Group Website

Western Blotting: Most Used Affinity Separation

Affinity and size information

Ubiquitous (> 50,000 citations)

Reliable

- Slow, manual operation

- Difficult for multiple proteins

- Lack of Chip or CE equivalent

Towbin, H., et al. Proc. Natl. Acad. Sci. U. S. A. 1979, 76 (9), 4350-4354.

Burnette, W. N., Anal. Biochem. 1981, 112 (2), 195-203.

http://www.cellsignal.com/products/9946.html

Traditional Western Blot WorkflowSeparate SDS-protein

complexes (~1-3 hours)

Target proteins

Antibody

specific

for target

Target proteins

Blocking proteins

Block (30 min)Detection

Immunoreaction

(hours)

Membrane

Blot (30-60 min)

Gel Filter

paper

Recent Micro Western Developments

O'Neill, R. A, et al. PNAS. 2006, 103 (44), 16153-16158.

Photo X-link

Electrode array

Hughs, A. J.; Herr, A. E. PNAS, 2012, 109, 21450–21455

Fast Sieving Separations Possible in Chips with Entangled Polymer Media

Bousse, L., Mouradian, S., Minalla, A., Yee, H., Williams,

K., Dubrow, R., Anal. Chem. 2001, 73, 1207–1212.

Chip Based Western Blot Separation field: 460 V/cm

Separation time: 2 min

Necessary to maintain

stable current

Contains MeOH:

Promote

adsorption

70

100

130

160

Gra

y V

alu

e

30 s

4 mm

Resolution of ERK1/2 (42/44kDa) from Cell Lysate on Membrane

On-column

In sheath

On membrane

Analysis of Multiple Proteins: “Stripping Analysis” in Conventional

Western• After 1st Westernremove antibody

• Apply 2nd Antibody

• Limited because of protein loss and time

www.piercenet.com

Multiple Separation Tracks on One Membrane (processed in parallel)

-Use repeated injection

from 1 sample: multi-

protein analysis

-Inject and deposit from

multiple samples

-Process all

immunoassays at 1 time

MEK1/2

Inj 1 Inj 2

ERK1/2

Inj 1 Inj 2

P-STAT3 STAT3

Inj 1 Inj 1

P-ERK1/2Inj 1 Inj 2

5 mm

11 kDa

21 kDa

32 kDa

40 kDa

65 kDa

9 proteins, n = 2800 nL sample

B-tubulin P-akt Akt P-MEK1/2

Inj 1 Inj 2 Inj 1 Inj 2 Inj 1 Inj 2Inj 1

11 kDa

21 kDa

32 kDa

40 kDa

65 kDa

Multiplexed Assay

Microfluidic Western Blot Summary:High Throughput, High Content

Ladder

Egg white

INS-1

10 kDa

40 kDa65 kDa

Ladder Lysozyme AMPK

Sloan-Kettering Institute, www.mskcc.org

High Throughput Screening (HTS)

• 100,000 to 2,000,000 candidates for one target

• High density well plates / Robots / Optical Readout

Problems:

-Assay development

-False Signals

-Reagent use:

Cost: $50k/screen

(50 mL7.5 L for 150,000 samples)

Difficult to express targets

Electrophoresis for Screening: Fast Assays

• -Enzyme Assays

• -GPCR activation

(Anal Chem, 2007,

79:1158)

• -Protein-Protein Interaction

(Anal Chem 2013,

85:9824)

*LRRASLG *LRRApSLGPKA

ATP

Fast Sample Introduction: Caliper High Throughput “Sipper Chip”

• Reads 384 Well Plate in 80 minutes

• Vacuum flow through channel is compromise

Segmented Flow for High Throughput Sample Delivery

Immiscible

Fluid

Teflon

Tube

Extraction Capillary

Injection Cross

Droplet Extraction for Electrophoretic Analysis

R R R R RR R R R R

S S S S S S S S S S

P P P P P

PP P P

P

Compound A5 Compound A6

Droplet MCE Screen: Sirt5 against Prestwick Library

• 384 samples

• 7 injections each

• 12 min

Comparison to LabChip SystemCaliper LabChip (1 sample x 1 injection)

Droplet-CE (75 samples x 8 replicates = 600 injections)

DyeProduct Substrate

Miniaturization of Screening

Assays in 96, 384, and 1536 well plates

Higher density have lower volume and use less reagent (saves costs for large scale screens)

Going smaller than 1536 (1 uL) is difficult: evaporation, liquid handling

Miniaturization: All droplet assay

Protein Protein Interaction as Drug Targets

Long believed to be

“undruggable”

Large number of

emerging targets

Small molecule

modulators

identified recently

• Protein–Protein Interactions as New Drug Targets, Enno Klussmann, John Scott,

Springer-Verlag Berlin Heidelberg, 2008.

• Aloy P, Russell RB, FEBS Lett. 2005 Mar 21;579(8):1854-8.

Part of human interactome

Protein-Protein Interaction Assays and Screens

• Surface Plasmon Resonance

• FRET

• Fluorescence Polarization

• Isothermal Calorimetry

• Bead & surface binding assays

Ideal: Fast, low sample consumption, label free,

easy to adapt to new proteins, multi-protein

complexes

Free* Complex

Affinity Probe CE(Noncompetitive Affinity Assay)

time

Antibody-Antigen

Aptamer-target

Protein-peptide

Ligand-receptor

DNA-protein

Drug-apoenzyme

Chaperone Protein-Protein Interactions

Hsp70-Bag

Hsp90 dimer

HOP

Hsp90 dimer

Hsp70

substrate

• Chaperone

interactions modulate

client protein binding

• Proteostasis

• Cancer/Protein

folding diseases

M. P. Mayer and B. Bukau, CMLS, Cell. Mol. Life Sci. Vol. 62, 2005

CE Assay for Protein-Protein Interaction

Current Standard Screen: Flow Cytometry Protein Interaction Assay

Hsp70

Bag3

Biotinylated Hsp70

Streptavidin/Biotin linkage

AF488 label

Bead Associated

Fluorescence

Measured by

Flow Cytometer

David L. Roman, et al, Mol Pharmacol 71:169–175, 2007

Streptavidin

Bead

Comparison of CE and Bead Assay

3,443 Compounds

Screened at 20 mM

118 Hits (3.4%)

69% confirmed 50% confirmed

100% reconfirm on FPCIA 50% reconfirm on CE

BeadCE

48 Hits (1.4%)

Dose-Response

Curve

Cross Platform

Test

Initial Screen

Conclusion: CE is More Selective

Why is CE More Selective?

Polymer

Bead Hsp70

Bag3

AF488 label

Bead Assay

CE Assay

Hsp70

Bag3

Hsp70

0 1 2 3

0

200

400

Time (min)

0

2

4

6

RF

U

0

30

60

Complex

Free

Calcein

Protein signal

gone

particles

Free

Complex

Fluorescent

compound

Aggregation

agent

Normal

compound

Hsp70

0 1 2 30

50

100

time (min)

RF

U

+ Bag3- Bag3

free Hsp70

?

Challenges for PPI Analysis by CE

Complex dissociation

Rauch, J.N., et al., Anal. Chem. 2013.

dissociationbridge

free

complex

30time (min)

RFU

Simultaneous: separations and interactions

Adsorption to Capillary

0 5 10

0

200

400

time (min)

RF

U

free

complex

free

complex

Protein Cross-linking Capillary Electrophoresis (PXCE)

quench

and

denature

+

gel electrophoresis

Protein Cross-linking CE

Chen, X. et al, J. Chromatogr. B. 2009.

RF

U

time (s) 480

400

mAb monomer

dimer

Therapeutic mAb

aggregation

0 5 100

150

300

time (min)

RF

U

free

Hsp70Hsp70-Bag3

6 8 10

0

9

18

time (min)

RF

U

free

Hsp90Hsp90

dimer

0 2 40

1000

2000

time (min)

RF

U

free

lysozyme

lysozyme-mab

6 9 120

5

10

time (min)

RF

U

free Hsp90

Hsp90

dimer2Hsp90-

FKBP52

Complexes Analyzed by PXCE

Bag

Hsp70

Effect of Hsp70-Bag3 Binding Site Mutants Detected by PXCE

Sondermann, H., et al. Science. 2001.

-10 -8 -6 -4

0

50

100

log(Hsp70 conc.)

perc

en

t in

hib

itio

n (

%)

WT

Hsp70 R,R 258,262, A,AHsp70 E,D 283,292 A,A

-9.0 -8.0 -7.0 -6.0 -5.0-9.0

-8.0

-7.0

-6.0

-5.0

log(Ki) by FCPIA

lo

g(K

i) b

y P

XC

E

Wild-Type

E,D 283,292 A,A

R,R 258,262 A,A

-10 -8 -6 -4

0

50

100

log(Hsp70 conc.)

perc

en

t in

hib

itio

n (

%)

WT

Hsp70 R,R 258,262, A,AHsp70 E,D 283,292 A,A

-9.0 -8.0 -7.0 -6.0 -5.0-9.0

-8.0

-7.0

-6.0

-5.0

log(Ki) by FCPIA

lo

g(K

i) b

y P

XC

E

Wild-Type

E,D 283,292 A,A

R,R 258,262 A,A

Competitive Inhibition by PXCE: Hsp70-Bag3 mutants

Quantification of Small Molecule Inhibition by PXCE

-10 -8 -6 -4

0

50

100

log(compound)

perc

en

t in

hib

itio

n (

%)

JG-231JG-98JG-311

JG-3

11

JG-9

8

JG-2

314

5

6

7

8

-lo

g(I

C50)

PXCE

FCPIA

Fast CE for “Sensing”

Fast CE

Aliquot Processing

Sampling Probe

Sample: Rxn, In vivo, Tissue

Time Resolved

Chemical

Information

“Sensing” Metabolites in the Living Brain:Identify chemical signals in behavior, learning, pharmacology,

pathophysiology

Rapid chemical changes Spatially heterogeneous

Delicate tissue

>100 neurotransmitters+ metabolites

Freely moving animals

Challenges:

aCSF

Pump Fractions

2.0

1.5

1.0

0.5

20151050

Microdialysis Sampling for In Vivo Monitoring

Microdialysis Probe

Sample transfer

tubing

-Highly Versatile but…

-Low Temporal Resolution

when coupled to HPLC

because of large sample

requirements (10 min)

aCSF

4 cm

PDMS Headpiece

Volume ~2 nL

On-line analysis

Pump

Collection Tubing

Off-line Analysis

Droplet Fraction Collection

MS

Enzyme

CE

MS

Enzyme

CE

-collect seconds (nL) fractions

-analyze on-line or off-line

-minimal distortion due to flow

-CE compatible

On-Board Droplet Generator for Awake Animal Experiments with Reagent Addition

Dialysis

Probe

Droplet

Generator

0.36

0.32

0.28

0.24

0.20

0.16

0.12

RF

U6005004003002001000

Relative Time (s)

100 s*

Monitoring by Fast CE

0.24

0.20

0.16

0.12

0.08

0.04

0.00

RF

U

50403020100Migration Time (s)

Electrophoresis Trace

* Time of analysis, in vivo time = 20 s

Applications

Effect of drugs on the brain

Changes in transmitters

with diseases

Changes in transmitters

with behavior

Changes in transmitters

with learning and memory

Huntington’s Disease

“CAG Repeat” disease

Neurodegenerative

Cognitive and motor

impairment

Death within 15 years

~30k cases in USA

Little treatment available

Glutamate Neurotransmission

Impaired glutamate uptake

Glu toxicity?

Recovery of Normal Glu Uptake in HD Mice with Cef

40

100

160

220

Perc

en

t Basal (

Glu

)

Series1

Series2

Series3

WT HD HD + Cef

80 s

Ceftriaxone Improves Motor Performance

Summary

Fast, Multiplexed Western

Droplet samples for high throughput

Nanoliter lab

PPI by CE gives high selectivity

PXCE for troublesome proteins

Fast CE for “sensing” in complex

environments

Acknowledgements