Cleavage sites and binding affinities

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS

BiC BioCentrum-DTUTechnical University of Denmark

Cleavage sites and binding affinities

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


Cleavage measurements

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


Peptide Digest Workflow

Purification of Peptides by RP-HPLC:Synthesized peptides are polished to >98% purity (1h

Gradient, 25 cm x 4 mm C18 column), lyophilized and used for digestion

Digestion: 100 ul Volume - 15 ul/ TimepointMSMS-Analysisvery fast – analysis of peptide digests can be performed in one

day multiple time points possible (Instrument time: 90 min/timepoint)Data Processing:Waters Protein Expression System (1-2h processing)Excel-Macros (Manual, 30 min/timepoint)

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


Peptide polishing:

very high purity of peptide substrates required, but some peptides ordered at >80% purity

are quite „dirty“ -> time consuming „polishing“ of peptides (4-8h)

Protocol has been established for routine purification

high hydrophobicity of some peptides leads to solubility /purification problems

Different/modified selection criteria for next QBC set?

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


Expression Data Acquisition

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


MHC Binding

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


RMAS Assay: classical way to measure peptide binding- However not quantitative (no determination of the affinity)

TAP difficient cell line

At 37 °C At 26 °C

Add peptide

Measure T cell activation

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


Experimental description of peptide-MHC binding

Hans-GeorgRammensee et al.,

www.syfpeithi.de

Søren Buus et al.,

www.cbs.dtu.dk/services/NetMHC/

How to examine HLA specificity?

”What the HLA has bound in vivo” Elution and sequencing of natural ligands

Simpel motif ~ low sensitivity predictions

”What the HLA will, or will not, bind in vitro” Determine the binding strength of any peptide

Extended motif ~ higher sensitivity predictions

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


How to determine peptide affinityHow to determine peptide affinityLaw of mass action

[R] + [L] [RL]

koff

kon

KD = koff (S-1)/kon (M-1S-1)

Peptide [M]

Bin

ding

KD = (10-15-10-6 M)

100%

50%

Peptide Log [M] Cold Peptide Log [M]

Log IC50B

ind i

n g h

o t p

eptid

e

Bi n

ding

[MHC] + [P] [P*MHC]

koff

kon

Saturation assay

Inhibition assay

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


How to do radioactive biochemical inhibition binding assays

• Obtain purified HLA

• Or recombinant heavy chain & b2m

• Obtain indicator peptide

• Perform dose titration of any inhibitory peptide

• Separate free from bound peptide

• Calculate binding and IC50

Non binding test peptide

Binding test Peptide

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


A spun column binding assayA spun column binding assay

G50

b2m

MHC

peptide

Binding test peptid

Non binding test peptid

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


PROSPROS

• Truly quantitativeTruly quantitative

• Can address affinities in Can address affinities in

the low nM levelthe low nM level

• ReproducibleReproducible

CONS

• Radioactive

• Not a standard method

• Waste problem

The radioactive biochemical binding assay

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


The Quantitative ELISA Capable of Determining Peptide-MHC Class I Interaction

Made possible by our recent development of highly active recombinant MHC class I heavy chains– functional equivalents of ”empty” molecules

Pros:• Reasonably simple, sensitive and quantitative• Does not depend on labeled peptide• It is easily adaptable to other laboratories

Disseminated protocol and standard reagents

L.O.Pedersen et al., , EJI. 2001, 31: 2986

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


DevelopmentDevelopment

Sensitivity below 0.1 nM or 5 x 10Sensitivity below 0.1 nM or 5 x 10-15-15 M M MHC class I complex !MHC class I complex !

Strategy for the assay

Step II: Detection of de novo folded MHC class I molecules by ELISA

IncubationIncubation

Step I: Folding of MHC class I molecules in solution

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


nM c

ompl

ex d

etec

ted

nM peptide offered

Results are expressed as:BMAX : Amount of detected complex

including 95% confidence interval

ELISA driven assay

KD: Peptide affinityincluding 95% confidence interval

Concentrations of complexes generated are plotted as a function of the concentration of peptide offered

Sylvester-Hvid, C. et al., Tissue Antigens (2002) 59:251

Automated, 384 format

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


Pros– Homologous proximity assay.– No washing => fast development

AlphaScreen

Acceptor bead

Donor bead

<200 nmO2•

Cons– Expensive reagents– Specialized reader

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


560 nM

480 nM

Biotin

Streptavidin

anti-mouse IgG

W6/32 mouse anti-HLA

AlphaScreen

O2•

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


AlphaScreen

0

1

10

100

1.000

10.000

100.000

0 1 10 100 1.000 10.000 100.000

ELISA KD nM

Alp

haS

cree

n K

D n

M

CE

NT

ER

FO

R B

IOL

OG

ICA

L S

EQ

UE

NC

E A

NA

LY

SIS


TAP Binding

• TAP is very hard to purify for in vitro assays

• Most used assay is the radioactive RIA assay

• IC50 from RIA assays are dependent of the affinity of the competing peptide

Documents

Cleavage sites and binding affinities