Protein Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry: Epitope Mapping and Higher Order Structure

1. 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
2

3. Exchange Rates of Hydrogens in Protein
O
O
H
N
H2N
O
OH
H
N
N
H
OH
O
OH
NH2
O
Hydrogen
Exchange Rate
OH, SH, NH2, CO2H, CONH2
fast
Main chain CONH
medium
Aliphatic CH
Aromatic CH
slow
3

4. H/D-Exchange-MS Analysis of Protein
H  D Exchange
H+, 0 °C
Protein
H/D-Exchange
for a certain
period of time
Amide hydrogens in
disordered regions
exchange fast
Virtually quench
exchange reaction by
shifting to low pH at low
temperature
Generate peptide
fragments to sublocalize deuterons
MS
H/D-Exchange is measured
as m/z shift by MS
Measure the m/z
of each peptide
Proteolysis
D2O incubation
HPLC
Spread out peptides
in chromatogram
4

5. What Does H/D-Exchange Rate Mean?
Can be calculated
We measure this
∆Gch‡
∆Gch‡ = – RT ln kch
N
H
N
D
∆Gex‡ = – RT ln kex
∆Gex‡
∆Gf = ∆Gex‡ – ∆Gch‡
= – RT ln (kex / kch)
∆Gf
N
H
N
D
O
O
5

6. H/D-Exchange-MS Analysis of Protein
H  D Exchange
Freeze Reaction
D2O incubation
Proteolysis
Protein Target
Several Time Points
m/z
H/D exchange is measured as
m/z shift by MS
Log (Time)
N
Sequence
fast
LC-MS
Intensity
Deuterium
Incorporation
Amide hydrogens in disordered
regions exchange fast
C
slow
fast
Identify slow-exchanging ordered regions and
fast-exchanging disordered regions
slow
Exchange Time
30 sec
100 sec
300 sec
1000 sec
3000 sec
10000 sec
30000 sec
100000 sec
Deuteration
Level
< 10%
> 10%
> 20%
> 30%
> 40%
> 50%
> 60%
> 70%
> 80%
> 90%
6

7. H/D-Exchange-MS Practice
 Automated data generation system and automated data analysis
software are required for efficient H/D-Exchange-MS practice
 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
Practice of H/D-Exchange-MS
ExSAR has in-house-developed data analysis software
7

8. Protein Characterization
by H/D-Exchange-MS
 How H/D-Exchange-MS Works
 Higher Order Structure Characterization
 Epitope Mapping
8

9. 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
9

10. Free Energy Change of hGH upon Folding
HDX-MS data
HDX rate
Free Energy
> - 2 kcal/mol
< - 2 kcal/mol
< - 4 kcal/mol
< - 6 kcal/mol
< - 8 kcal/mol
10

11. 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
11

12. pH Dependent Stability of hGH
pH 2.7
pH 4.4
> - 2 kcal/mol
< - 2 kcal/mol
< - 4 kcal/mol
< - 6 kcal/mol
< - 8 kcal/mol
pH 5.9
pH 7.5
12

13. 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
13

14. Protein Characterization
by H/D-Exchange-MS
 How H/D-Exchange-MS Works
 Higher Order Structure Characterization
 Epitope Mapping
14

15. Best Selling Drugs in 2010
Brands®
Lipitor
Plavix
Enbrel*
Advair
Remicade**
Avastin**
Rituxan**
Abilify
Diovan
Companies
Pfizer, Astellas
BMS, Sanofi Aventis
Amgen, Pfizer Takeda
Glaxo Smith Kline
J&J, Merck, Mitsubishi Tanabe
Roche
Roche
Otsuka, BMS
Novartis
Indications
Cholesterol
Atherosclerosis
Arthritis
Asthma
Arthritis
Colon cancer
Non Hodgkin’s Lymphoma
Schizophrenia
Hypertension
Crestor
Astra Zeneca, Shionogi
Cholesterol
5.6
Humira**
Abbott
Arthritis
5.4
Herceptin**
Roche
Breast Cancer
5.1
* fusion protein
** monoclonal antibody
$ billion
11.4
9.6
8.4
7.8
7.4
6.9
6.5
6.2
6.1
http://knol.google.com/k/krishan-maggon/top-ten-twenty-best-selling-drugs-2010/3fy5eowy8suq3/141#
Therapeutic antibody is the fastest growing sector in pharmaceutical industry
15

16. 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
16

17. 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
Nemeth, Centocor, GEN Webinar April 29, 2009
17

18. Reliable
X-ray
NMR
Mutagenesis
HDX-MS
Less Reliable
Low Resolution
High Resolution
Epitope Mapping Methods
compatible with
conformational
epitope
Pepscan
proteolysis
Phage
Low throughput
High throughput
Low applicability
High cost
High applicability
Low cost
18

19. On-Exchange for Epitope Mapping
<On-solution without antibody>
D2O
[H+]
digestion
HPLC
antigen
<On-solution with antibody>
H2O
D2O
[H+]
digestion
HPLC
antibody
Coales et al., RCM 2009, 23, 639-647
19

20. On/Off-Exchange for Epitope Mapping
<Labeling Experiment --- on-solution/off-column>
D2O
D2O
[H+]
H2O
digestion
HPLC
antigen-antibody
complex
antigen
<Control Experiment --- on-column/off-column>
H2O
D2O
H2O
[H+]
digestion
HPLC
antibody in a column
Coales et al., RCM 2009, 23, 639-647
20

21. 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
21

22. H/D-Exchange Epitope Mapping: IL-17A
weak H/D-Exchange protection
strong H/D-Exchange protection
Gerhardt et al., JMB, 2009, 394, 905
22

23. IL-17A – Epitope Mapping
Putative epitopes mapped onto IL-17F structure
peptide
• One must be right and one wrong!
• How do we resolve the discrepancy?
H/D-Exchange
Gerhardt et al., JMB, 2009, 394, 905
23

24. IL-17A – Crystal Structure
in Complex with Fab
Gerhardt et al., JMB, 2009, 394, 905
24

25. Epitope Mapping: Cytochrome C – E8 Antibody
10
20
30
40
GDV EKGKK I F VQKCAQCH T V EKGGKHK TGPN L HG L FGRK TGQAPG F T
♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦
●●● ● ●●● ●●
● ●● ●●●
●
50
60
70
80
90
100
Y T DANKNKG I TWKE E T LME Y L ENPKKY I PG T KM I F AG I KKK T ERED L I AY L KKA T NE
♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦
●●
●●●● ●●
●●
●
●●
●●●
X-ray crystallography defined contact residue ( ≥ 10 Å2)
H/D-Exchange-MSMS defined epitope (protected ≥ 10%)
Coales et al., RCM 2009, 23, 639-647
25

26. Combination of H/D-Exchange-MS and
Docking for Epitope Mapping
H/D-Exchange-MS Positive
False positive due to
allosteric effects and
medium resolution
Docking Positive
False positive mostly due to
not perfect energy function
and induced fit
Most probably right answer
26

27. Docking of Cyt-c and E8
without H/D-Exchange-MS Constraints
Non-CDR
blocked
E8 Antibody
(1QBL)
Docking
Compare
Cytochrome c
(1HRC)
Co-crystal
(1WEJ)
Pandit et al., JMR in press
27

28. Docking of Cyt-c and E8
without H/D-Exchange-MS Constraints
Red
X-ray
Cytochrome c
Pandit et al., JMR in press
28

29. Epitope Mapping: Cytochrome C – E8 Antibody
10
20
30
40
GDV EKGKK I F VQKCAQCH T V EKGGKHK TGPN L HG L FGRK TGQAPG F T
♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦
●●● ● ●●● ●●
● ●● ●●●
●
50
60
70
80
90
100
Y T DANKNKG I TWKE E T LME Y L ENPKKY I PG T KM I F AG I KKK T ERED L I AY L KKA T NE
♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦
●●
●●●● ●●
●●
●
●●
●●●
X-ray crystallography defined contact residue ( ≥ 10 Å2)
H/D-Exchange-MS defined epitope (protected ≥ 10%)
Coales et al., RCM 2009, 23, 639-647
29

30. Docking of Cyt-c and E8
with H/D-Exchange-MS Constraints
Non-CDR
blocked
E8 Antibody
(1QBL)
Docking
Compare
Non-H/D perturbed
blocked
Cytochrome c
(1HRC)
Co-crystal
(1WEJ)
Pandit et al., JMR in press
30

31. Docking of Cyt-c and E8
without H/D-Exchange-MS Constraints
Red
X-ray
Cytochrome c
Pandit et al., JMR in press
31

32. Docking of Cyt-c and E8
with H/D-Exchange-MS Constraints
Blue
Top pose
Docking
Red
X-ray
Cytochrome c
Pandit et al., JMR in press
32

33. Reliable
X-ray
NMR
Mutagenesis
H/D-Exchange-DOCK
+ Computation
H/D-Exchange-MS
Less reliable
Low Resolution
High Resolution
Epitope Mapping Methods
compatible with
conformational
epitope
Pepscan
proteolysis
Phage
Low throughput
High throughput
Low applicability
High cost
High applicability
Low cost
33

34. Epitope Mapping of TLR3 by H/D-Exchange-MS
mAb A
mAb B
H/D-Exchange-MS defined epitope (protected ≥ 10%)
Pomerantz et al., 59th Annual ASMS Conference, Poster TP485
34

35. 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
35

36. 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
36

37. Expanding the Utility of a Commercial
HDX Automation Solution
Eric B. Monroe
University of Arizona

38. Retroviral Assembly and Maturation
Bacteriophage
Briggs, PNAS 27:106 (2009)
Prevelige Lab
Tang et al. Structure 19:496 (2011)
Nanomaterials
80α
SAPI 1 Size
Determination
(Dokland Lab)
SAPI 1

39. Challenges to Our Structure MS Projects
Nonexpert Usage
Reproducibility
Randomization
Temperature Control
Modulation of Ex1 Exchange/Protein Dynamics
Solution Phase Digestion
Stability Assays

40. Challenges to Our Structure MS Projects
Nonexpert Usage
Reproducibility
Randomization
Temperature Control
Modulation Of Ex1 Exchange/Protein Dynamics
Solution Phase Digestion
Stability Assays

41. Automation Solution from LEAP
4 °C
iso
pepsin
trap
column
LC
20 °C
4 °C
sample
exchange
quench
to MS

42. Flexibility of the LEAP Hardware
4 °C
iso
trap
column
LC
20 °C
4 °C
to MS

43. Flexibility of the LEAP Hardware
4 °C
iso
trap
column
LC
20 °C
4 °C
exchange
buffers
quench
exchange
digestion
to MS

44. Software Settings Allow Rapid
Modification of Experiments

45. 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

46. Automation Greatly Improves Reproducibility of
Exchange Measurements
Intact protein
A
1-32 6+
12+
A
B
B
C
C
SAPI1
His6-gp6
15 min exchange
33.14 ± 0.18 Dincorp
RSD of 0.54%
2 min exchange
2 min solution digest (pepsin)
Manual preps
RSD ~5-10%
7.20 ± 0.08 Dincorp
RSD 1.11%

47. Samples Held at Stable Temperatures
(Selectable and Controllable) throughout Experiment
exchange plate
quench plate
PCR coolers for exchange/quench
Very stable at multiple temperatures (±0.1 °C over >5 min)
• Often sample limited (particularly with VLPs and assemblies)
• Necessitates small volumes to minimize losses
• Drawers temperature controlled and PCR coolers keep very stable temperature

48. Exploiting Temperature Control to
Examine Protein Dynamics
45s exchange
10+
Engen, Curr Protocols (2009)
Change in temperature can
modulate EX1/EX2 exchange activity

49. 11+
EX2
20 °C
11+
4 °C
EX1
0.25 min
0.5 min
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
1 min
1.5 min
2.5 min
3.5 min
5 min

50. Expanding Digestion Options
• Standard concept utilizes immobilized pepsin
• Poor/incomplete digest of some proteins
• Others contained coverage gaps in important regions
20 °C
D2O
protein exchange
2 °C
quench
protease

51. Solution Digests
• Minimal increase in back exchange over column digests
• Added flexibility to improve coverage
pepsin
Center of 4-helix bundle
XIII
several critical residues by mutagenesis
pepsin
XIII
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

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

53. Pseudo-SUPREX Automation
Standard results:
Monroe, Structure (2010) 18, 1483-1491
Oas, PNAS (2000), 8296-8301
• 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

54. Pseudo-SUPREX Automation
Standard results:
20 °C
D2O +
GuHCl
protein
exchange
2 °C
quench
protease
Oas, PNAS (2000), 8296-8301
• 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

55. 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

56. Examining HIV CA Assemblies with
Pseudo-SUPREX
• Peptide spanning H1-H2 shows similar
CA 23-40 3+ properties in monomer
2.5 min of exchange

57. Examining HIV CA Assemblies with
Pseudo-SUPREX
• But variation observed between monomeric and
CA 23-40 3+ assembled samples
2.5 min of exchange
Suggests
intermolecular
interface
greatly
strengthens
subunit
structural
fidelity
Peptides not
involved in
intermolecular
interfaces are
also stabilized

58. Flexibility is the key to these experiments.
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.
Various temp
2 °C
Randomization of samples, modulation
EX1 exchange/protein dynamics,
solution phase digestion, SUPREX–like
stability assays.

59. Acknowledgements
Peter Prevelige
David Morris
James Cherwa
Rui Li
Terje Dokland
Altaira Dearborn
Michael Spilman
Peter Smith
Matt Renfrow
LeeAnn Boerma
National Institutes of Health
RR17261, DK077279-01,
R01AI044626, F32GM087994