Chromatography: Protein Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry

Protein Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry:

Epitope Mapping and Higher Order Structure

Yoshitomo Hamuro

2

Protein Characterization by H/D-Exchange-MS

How H/D-Exchange-MS Works Higher Order Structure Characterization Epitope Mapping


3

Exchange Rates of Hydrogens in Protein

H2N

O O

O O

OH NH

HN

HN

OH

O

OH

NH2

Hydrogen Exchange Rate OH, SH, NH2, CO2H, CONH2 fast Main chain CONH medium Aliphatic CH Aromatic CH slow

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H/D-Exchange-MS Analysis of Protein

H D Exchange

D2O incubation

Protein

H+, 0 °C

Amide hydrogens in disordered regions

exchange fast

H/D-Exchange is measured as m/z shift by MS

Virtually quench exchange reaction by

shifting to low pH at low temperature

H/D-Exchange for a certain

period of time

Generate peptide fragments to sub- localize deuterons

Prot

eoly

sis

HPLC

Spread out peptides in chromatogram

MS

Measure the m/z of each peptide

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What Does H/D-Exchange Rate Mean?

∆Gf

∆Gch‡

∆Gch‡ = – RT ln kch

∆Gex

‡ = – RT ln kex ∆Gf = ∆Gex

‡ – ∆Gch‡

= – RT ln (kex / kch)

∆Gex‡

NH

O

ND

O

NH

ND

Can be calculated

We measure this

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H/D-Exchange-MS Analysis of Protein

H D Exchange

D2O incubation

Protein Target

Freeze Reaction

Proteolysis

LC-MS Amide hydrogens in disordered regions exchange fast

H/D exchange is measured as m/z shift by MS

Inte

nsity

m/z

Exchange Time 30 sec 100 sec 300 sec 1000 sec 3000 sec 10000 sec 30000 sec 100000 sec

< 10% > 10% > 20% > 30% > 40% > 50% > 60% > 70% > 80% > 90%

Deuteration Level

Several Time Points

Sequence N C

Deut

eriu

m

Inco

rpor

atio

n

Log (Time)

Identify slow-exchanging ordered regions and fast-exchanging disordered regions

slow slow fast fast

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H/D-Exchange-MS Practice

Automated data generation system and automated data analysis software are required for efficient H/D-Exchange-MS practice

Practice of H/D-Exchange-MS

Experimental sequence of events controlled in a timely manner at low temperature Each project requires different data acquisition and poses a

different set of problems. ExSAR uses a modular approach to allow adaptability for

project dependent experimental and data acquisition conditions not available from a standalone platform

Automated data analysis software Data analysis is the most time consuming step ExSAR has in-house-developed data analysis software

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H/D-Exchange-MS Results of Human Growth Hormone (hGH)

Exchange Time

30 sec 100 sec 300 sec 1000 sec 3000 sec 10000 sec 30000 sec 100000 sec

Hamuro et al., J. Biomol. Techniques 2003, 14, 171

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Free Energy Change of hGH upon Folding

> - 2 kcal/mol < - 2 kcal/mol < - 4 kcal/mol < - 6 kcal/mol < - 8 kcal/mol

HDX-MS data

HDX rate

Free Energy

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H/D-Exchange-MS Results of hGH at Four Different pHs

pH

2.7

4.4

5.9

7.5

H/D-Exchange-MS can characterize protein samples in various conditions

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pH Dependent Stability of hGH

> - 2 kcal/mol < - 2 kcal/mol < - 4 kcal/mol < - 6 kcal/mol < - 8 kcal/mol

pH 2.7 pH 4.4

pH 5.9 pH 7.5

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Higher Order Structure Characterization by H/D-Exchange-MS

H/D-Exchange-MS is a powerful technology used to characterize a protein’s higher order structure in solution

H/D-Exchange-MS widely applicable medium resolution medium throughput

Potential applications include formulation optimization quality control Biosimilar

Companies have started using H/D-Exchange-MS data as higher order structure characterization of protein therapeutics for regulatory agency filing

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http://knol.google.com/k/krishan-maggon/top-ten-twenty-best-selling-drugs-2010/3fy5eowy8suq3/141#

Best Selling Drugs in 2010

Brands® Companies Indications $ billion Lipitor Pfizer, Astellas Cholesterol 11.4 Plavix BMS, Sanofi Aventis Atherosclerosis 9.6 Enbrel* Amgen, Pfizer Takeda Arthritis 8.4 Advair Glaxo Smith Kline Asthma 7.8 Remicade** J&J, Merck, Mitsubishi Tanabe Arthritis 7.4 Avastin** Roche Colon cancer 6.9 Rituxan** Roche Non Hodgkin’s Lymphoma 6.5 Abilify Otsuka, BMS Schizophrenia 6.2 Diovan Novartis Hypertension 6.1 Crestor Astra Zeneca, Shionogi Cholesterol 5.6 Humira** Abbott Arthritis 5.4 Herceptin** Roche Breast Cancer 5.1

Brands® Companies Indications $ billion Lipitor Pfizer, Astellas Cholesterol 11.4 Plavix BMS, Sanofi Aventis Atherosclerosis 9.6 Enbrel* Amgen, Pfizer Takeda Arthritis 8.4 Advair Glaxo Smith Kline Asthma 7.8 Remicade** J&J, Merck, Mitsubishi Tanabe Arthritis 7.4 Avastin** Roche Colon cancer 6.9 Rituxan** Roche Non Hodgkin’s Lymphoma 6.5 Abilify Otsuka, BMS Schizophrenia 6.2 Diovan Novartis Hypertension 6.1 Crestor Astra Zeneca, Shionogi Cholesterol 5.6 Humira** Abbott Arthritis 5.4 Herceptin** Roche Breast Cancer 5.1

* fusion protein ** monoclonal antibody

Therapeutic antibody is the fastest growing sector in pharmaceutical industry

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Why Epitope Mapping?

Scientific reason

Mechanism of action

IP reason

Protecting epitope is potential more powerful than protecting substance

Regulatory reason

FDA asks to identify epitope as much as possible

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Nemeth, Centocor, GEN Webinar April 29, 2009

Advantages and Challenges

Technologies / Preference Pros Cons

Crystallography1 Gold standard with high precision; works for both Le and Ce

Require high- quality Ab / Ag proteins; challenge for co-crystallization

NMR3 Done in solution; complementary to crystallography

Limited to size of proteins; time-consuming to make isotope-labeled proteins.

Mutagenesis1 Straight forward; identify key residues for epitopes

Highly rely on protein expression; folding properly is a concern

H/D-Exchange-MS1 Straight forward, moderate resolution; works for both Le and Ce

Challenge for glycosylated antigens

Protease digest / MS1 Easy to do; works well with Linear epitopes (Le)

Low resolution; Not working with Conformational epitopes (Ce)

PepScan2 (Overlapping peptides)

Fast to do; with residue-level resolution; membranes can be reused

Limited to linear epitopes; need good controls to discern false results

Phage peptide panning3 HTP analysis; parallel panning for different targets.

Limited to linear epitopes; may reveal mimetopes

Electron Microscopy2 consumes less amount of proteins Low resolution; needs protein structures to interpret the data

Computational modeling2 No need for proteins; predicting epitopes for further verification

requires both Ab/Ag structures; needs experimental validation

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Epitope Mapping Methods

High throughput

Low cost

Low throughput

High cost

High

Res

olut

ion

Relia

ble

Low

Res

olut

ion

Less

Rel

iabl

e X-ray

NMR Mutagenesis

Pepscan proteolysis

Phage

HDX-MS compatible with conformational

epitope

Low applicability High applicability

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On-Exchange for Epitope Mapping

<On-solution with antibody>

<On-solution without antibody>

H2O

antigen

D2O

[H+] digestion HPLC

D2O

[H+] digestion HPLC

antibody

Coales et al., RCM 2009, 23, 639-647

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On/Off-Exchange for Epitope Mapping

<Control Experiment --- on-column/off-column>

<Labeling Experiment --- on-solution/off-column>

H2O

antigen antigen-antibody complex

D2O

[H+] digestion HPLC

D2O H2O

[H+] digestion HPLC

H2O D2O

Coales et al., RCM 2009, 23, 639-647

antibody in a column

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H/D-Exchange Perturbation of IL-17A by Antibody

strongly H/D-Exchange protected by Ab weakly H/D-Exchange protected by Ab not protected

Gerhardt et al., JMB, 2009, 394, 905

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H/D-Exchange Epitope Mapping: IL-17A

strong H/D-Exchange protection

weak H/D-Exchange protection


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IL-17A – Epitope Mapping

• One must be right and one wrong! • How do we resolve the discrepancy?

Putative epitopes mapped onto IL-17F structure

peptide

H/D-Exchange


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IL-17A – Crystal Structure in Complex with Fab


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Epitope Mapping: Cytochrome C – E8 Antibody

X-ray crystallography defined contact residue ( ≥ 10 Å2) H/D-Exchange-MSMS defined epitope (protected ≥ 10%)

10 20 30 40GDVEKGKK I F VQKCAQCHT VEKGGKHKTGPNLHGL FGRKTGQAPGF T♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦●●● ● ●●● ●● ● ●● ●●● ●

50 60 70 80 90 100Y TDANKNKG I TWKEE T LMEY L ENPKKY I PGTKM I FAG I KKKT EREDL I AY LKKATNE♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦●● ●●●● ●● ●● ● ●● ●●●

Coales et al., RCM 2009, 23, 639-647

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Combination of H/D-Exchange-MS and Docking for Epitope Mapping

H/D-Exchange-MS Positive Docking Positive

False positive due to allosteric effects and medium resolution

False positive mostly due to not perfect energy function and induced fit

Most probably right answer

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Docking of Cyt-c and E8 without H/D-Exchange-MS Constraints

Non-CDR blocked

Cytochrome c (1HRC)

E8 Antibody (1QBL)

Co-crystal (1WEJ)

Compare

Docking

Pandit et al., JMR in press

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Cytochrome c

Red X-ray


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Epitope Mapping: Cytochrome C – E8 Antibody

X-ray crystallography defined contact residue ( ≥ 10 Å2) H/D-Exchange-MS defined epitope (protected ≥ 10%)

10 20 30 40GDVEKGKK I F VQKCAQCHT VEKGGKHKTGPNLHGL FGRKTGQAPGF T♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦●●● ● ●●● ●● ● ●● ●●● ●

50 60 70 80 90 100Y TDANKNKG I TWKEE T LMEY L ENPKKY I PGTKM I FAG I KKKT EREDL I AY LKKATNE♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦●● ●●●● ●● ●● ● ●● ●●●

Coales et al., RCM 2009, 23, 639-647

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Docking of Cyt-c and E8 with H/D-Exchange-MS Constraints

Non-CDR blocked

Non-H/D perturbed blocked

Docking

Cytochrome c (1HRC)

E8 Antibody (1QBL)

Co-crystal (1WEJ)

Compare


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Cytochrome c

Red X-ray


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Docking of Cyt-c and E8 with H/D-Exchange-MS Constraints

Blue Top pose Docking

Cytochrome c

Red X-ray


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Epitope Mapping Methods

High throughput

Low cost

Low throughput

High cost

High

Res

olut

ion

Relia

ble

Low

Res

olut

ion

Less

relia

ble

X-ray NMR

Mutagenesis

Pepscan proteolysis

Phage

H/D-Exchange-MS

+ Computation

H/D-Exchange-DOCK

compatible with conformational

epitope

Low applicability High applicability

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Epitope Mapping of TLR3 by H/D-Exchange-MS

H/D-Exchange-MS defined epitope (protected ≥ 10%)

mAb A mAb B

Pomerantz et al., 59th Annual ASMS Conference, Poster TP485

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Epitope Mapping by H/D-Exchange-MS

Therapeutic antibody is the fastest growing sector in pharmaceutical industry

Epitope mapping is a critical step for

Scientific reasons IP reasons Regulatory reasons

Epitope mapping by H/D-Exchange-MS is a powerful option wide-applicability medium resolution medium-throughput compatibility with discontinuous conformational epitopes compatibility with glycosylated and large proteins high resolution by combining with computational docking

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Acknowledgments

ExSAR Stephen J. Coales Kathleen S. Molnar Steven J. Tuske Kelly E Deepangi Pandit

Janssen Jennifer F. Nemeth

AstraZeneca Mark Abbott

UPenn Yvonne Paterson

Financial support NIH

Expanding the Utility of a Commercial HDX Automation Solution

Eric B. Monroe

University of Arizona

Prevelige Lab

Retroviral Assembly and Maturation

Nanomaterials

Bacteriophage

SAPI 1 Size Determination (Dokland Lab)

80α SAPI 1

Tang et al. Structure 19:496 (2011)

Briggs, PNAS 27:106 (2009)

Challenges to Our Structure MS Projects Nonexpert Usage

Reproducibility

Randomization

Temperature Control

Modulation of Ex1 Exchange/Protein Dynamics

Solution Phase Digestion

Stability Assays

Nonexpert Usage

Reproducibility

Randomization

Temperature Control

Modulation Of Ex1 Exchange/Protein Dynamics

Solution Phase Digestion

Stability Assays

Challenges to Our Structure MS Projects

Automation Solution from LEAP

20 °C 4 °C

exchange

sample

quench

4 °C

LC

iso

pepsin

trap

column to MS

Flexibility of the LEAP Hardware

20 °C 4 °C

4 °C

LC

iso

trap

column to MS

Flexibility of the LEAP Hardware

20 °C 4 °C

exchange

quench

digestion

4 °C

LC

iso

trap

column to MS

exchange buffers

Software Settings Allow Rapid Modification of Experiments

Tweaking Workflow for Randomization • Software “requires” runs in increasing time of exchange • Solution—import list and queue

multiple runs

Excel File

0, 15s, 30s, 1m, 2m, 4m, 8m, 15m, 30m, 60m runs in triplicate

Automation Greatly Improves Reproducibility of Exchange Measurements

12+

15 min exchange

33.14 ± 0.18 Dincorp RSD of 0.54%

2 min exchange 2 min solution digest (pepsin)

7.20 ± 0.08 Dincorp

RSD 1.11%

1-32 6+

Intact protein

A B C

A B C

SAPI1 His6-gp6

Manual preps RSD ~5-10%

Samples Held at Stable Temperatures (Selectable and Controllable) throughout Experiment

quench plate

exchange plate

PCR coolers for exchange/quench

• Often sample limited (particularly with VLPs and assemblies)

• Necessitates small volumes to minimize losses

• Drawers temperature controlled and PCR coolers keep very stable temperature

Very stable at multiple temperatures (±0.1 °C over >5 min)

Exploiting Temperature Control to Examine Protein Dynamics

45s exchange 10+

Change in temperature can modulate EX1/EX2 exchange activity

Engen, Curr Protocols (2009)

Exchange @ 20 °C and 4 °C in pD 6.98 and

7.51 D2O buffers respectively

11+ ions show change

in EX1 breathing mechanism of

HLH motif

Half-life of opening extended from ~75s

to ~130s

20 °C 4 °C

0.25 min

0.5 min

1 min

1.5 min

2.5 min

3.5 min

5 min

11+ 11+

EX1 EX2

Expanding Digestion Options • Standard concept utilizes immobilized pepsin • Poor/incomplete digest of some proteins • Others contained coverage gaps in important regions

D2O quench

protease protein exchange

2 °C 20 °C

pepsin

XIII

pepsin

XIII

Solution Digests • Minimal increase in back exchange over column digests • Added flexibility to improve coverage

Center of 4-helix bundle several critical residues by mutagenesis

500 ng protein (similar maps with 1.5 µg) 2 min digest with 15 min LC MS All mapped ions were able to be followed by HDX

Solution Digests • Minimal increase in back exchange over column digests • Added flexibility to improve coverage

500 ng protein (similar maps with 1.5 µg) 2 min digest with 15 min LC MS All mapped ions were able to be followed by HDX

pepsin

XIII

pepsin

XIII

Pseudo-SUPREX Automation

• Stability of Unpurified Proteins from Rates of H/D Exchange • Titration of GuHCl under same time of exchange, D2O

concentration and pD • To automate, D2O-GuHCl buffers placed in D2O plate and set

runs for same exchange time

Monroe, Structure (2010) 18, 1483-1491

Oas, PNAS (2000), 8296-8301

Standard results:

Pseudo-SUPREX Automation

• Stability of Unpurified Proteins from Rates of H/D Exchange • Titration of GuHCl under same time of exchange, D2O

concentration and pD • To automate, D2O-GuHCl buffers placed in D2O plate and set

runs for same exchange time

Oas, PNAS (2000), 8296-8301

Standard results:

D2O + GuHCl quench

protease protein exchange

2 °C 20 °C

HIV CA Protein by Pseudo-SUPREX

• CA does not undergo cooperative unfolding as could be expected as a multidomain protein

• Maintains ability to examine stability

What about with digest? Or in an assembled state?

2.5 min of exchange

Examining HIV CA Assemblies with Pseudo-SUPREX • Peptide spanning H1-H2 shows similar

properties in monomer CA 23-40 3+

2.5 min of exchange

• But variation observed between monomeric and assembled samples

CA 23-40 3+

Suggests intermolecular interface greatly strengthens subunit structural fidelity Peptides not involved in intermolecular interfaces are also stabilized

2.5 min of exchange

Examining HIV CA Assemblies with Pseudo-SUPREX

Flexibility is the key to these experiments.

Various temp 2 °C

Exploiting the flexibility of the autosampler allows for the

incorporation of multiple variations of HDX into lab workflows.

Temperature control, reproducibility, throughput, and ease of use allow

nonexpert usage.

Randomization of samples, modulation EX1 exchange/protein dynamics,

solution phase digestion, SUPREX–like stability assays.

Acknowledgements Peter Prevelige

David Morris James Cherwa

Rui Li

Matt Renfrow LeeAnn Boerma

Terje Dokland Altaira Dearborn Michael Spilman

Peter Smith

National Institutes of Health RR17261, DK077279-01,

R01AI044626, F32GM087994

Chromatography: Protein Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry

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Transcript of Chromatography: Protein Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry