Complementary study of two structural mass spectrometry methodologies

1
Complementary study of two structural mass
spectrometry methodologies
Synchrotron footprinting and hydrogen/deuterium
exchange mass spectrometry
Xiaojing Zheng
Supervisor Dr. Mark Chance
Case center for proteomics and Mass Spectrometry
01/31/2008
2
Background

• Synchrotron Footprinting Mass spectrometry
• H/D exchange Mass sepctrometry
• Serpin

3
1. What are the two structural MS methodologies
?
SFMS
HXMS
4
2. Advantages of structural MS methodologies

• high sensitivity (picomoles of sample are
  required)
• relatively fast analysis
• no practical size limitation
• provide protein solution conformation data under
  a variety of physiological conditions
• provide essential dynamics information that is
  not evident from the static crystal structure

5
3. Serpin

• SERine Proteinase INhibitors
• a superfamily of proteins ( gt 400)
• a single common core domain consisting of three
  ß-sheets and 8-9 a helices

Front
Back
Elliott R. et al, (1998) J. Mol. Biol., 275,419
6
Two pillars of protein folding
4. Why serpin?

• Interesting structural design and unique
  inhibition mechanism
• their critical roles in many highly regulated
  physiological processes

2) The native state is the most thermodynamically
stable
1) Sequence determines structure
ATGFDRRAAYLKSSEA
7
Proteins that break the rules

• Prions
• A few proteases
• Some viral proteins (e.g. hemagglutinin)
• Serpins

8
4. The serpin inhibitory mechanism suicide
RCL (Reactive center loop)
9
5. ?1-Antitrypsin

• most abundant serpin in human plasma (1-2 mg/mL)
• important role in inflammation major inhibitor
  of neutrophil elastase
• ?1AT deficiency (MIM 107400) is associated with
  early-onset emphysema
• and liver disease

10
Results

• H/D exchange data
• Footprinting data
• Comparison of structural MS data and
  crystallographic data
• Comparison of footprinting and H/D exchange
  consistent data conflicting data

11
1. Peptide mapping and coverage
H/D exchange
Footprinting
Protease Pepsin
Trypsin, Chymotrysin, Asp-N
Coverage 89
90
Gap Strand 2A
Helix C

• Comparable coverage on ?1 AT (394 aa, 44 kDa)
• Usage of multiple enzymes in footprinting
• 1) Maximize the sequence coverage
• 2) Increase the structural resolution
• 3) Provide redundant information on peptides
  with modified
• residues overlap

12
hA
s6B
hB

• 23 FNKITPNL AEFAFSLYRQ LAHQSNSTNI
  FFSPVSIATA FAMLSLGTKA 70
• DTHDEILEGL NFNLTEIPEA QIHEGFQELL HTLNQPDSQL
  QLTTGNGLFL 120
• 121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY
  VEKGTQGKIV 170
• 171 DLVKELDRDT VFALVNYIFF KGKWERPFEV KDTEEEDFHV
  DQVTTVKVPM 220
• 221 MKRLGMFNIQ HCKKLSSWVL LMKYLGNATA IFFLPDEGKL
  QHLENELTHD 270
• IITKFLENED RRSASLHLPK LSITGTYDLK
  SVLGQLGITK VFSNGADLSG 320
• VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA MFLEAIPMSI
  PPEVKFNKPF 370
• 371 VFLMIDQNTK SPLFMGKVVN PTQK 394

hD
hC
s2A
hF
hE
s1A
s3C
s3A
s4C
s3C
s1B
hG
hH
s2B
s3B
hH
hI
s6A
s2C
s5A
R.C.L.
s1C
s4B
s5B
Dotted line pepsin (H/D exchange) Black
trypsin purple chymotrypsin green Asp-N
13
2. H/D exchange data
14
5. H/D exchange data vs. crystallographic data
Front
Back
Nslow(experimental)
2
N 20 structure (X-ray)
10
15
3. Footprinting data
Chromatography of tryptic peptide 344-365
47.56
NL
100
3.50E6
(MH)2
344 GTEAAGAMFLEAIPMSIPPEVK 365
m/z
80
1129.50-
1130.70 F
60
MS 80ms
40
20
0
45.92
NL
100
3.58E6
344 GTEAAGAMFLEAIPMSIPPEVK 365
(MH16)2
m/z
80
344 GTEAAGAMFLEAIPMSIPPEVK 365
1137.50-
1138.70 F
60
MS 80ms
Relative Abundance
40
20
0
NL
44.89
100
5.06E6
(MH32)2
344 GTEAAGAMFLEAIPMSIPPEVK 365
m/z
80
1145.50-
1146.70 F
60
MS 80ms
40
20
0
32
34
36
38
40
42
44
46
48
50
52
54
Time (min)
16
Full spectra of unexposed and 100ms exposed
tryptic peptide 202-217
0 ms
(MH)3
(MH)2
632.21
100
90
947.02
80
70
60
Relative Abundance
50
40

30
(MH)1
20
10




1892.73
0
400
600
800
1000
1200
1400
1600
1800
2000
M/Z
100 ms
100
90
80
70

(MH)2
60
Relative Abundance

946.99
50
(MH)3
40
632.50
30

20
(MH)1
636.93
10
954.62
510.34
1892.68
0
400
600
800
1000
1200
1400
1600
1800
2000
M/Z

17
Tandem MS spectrum of tryptic peptide 291-300
b1 b2 b3 b4 b5 b6 b7 b8 b9
y9 y8 y7 y6 y5 y4 y3 y2 y1
18
Dose-response curve
Peptide 202-217

Peptide 301-310

1.2
1.1
1
0.9
0.8
0.7
0.6

Fraction Unmodified
0.5

0.4
0.3
0.2
0
10
20
30
40
50
60
Exposure time (ms)
19
Oxidation rate and modified residues of peptides
from ?1 antitrypsin
20
4. Footprinting data vs. crystallographic data

• Two criteria
• Solvent-accessibility
• 2) Reactivity Cys gt Met gt Trp gt Tyr gt Phe gt
  Cystine gt His gt Leu, Ile gt Arg , Lys,
• Val gt Pro, Ser, Thr gt Gln, Glu gt Asp, Asn gt Ala gt
  Gly
• Behavior of 14 oxidized and 11 unoxidized
  peptides were generally consistent with the SASA
  calculated on the ?1AT (1QLP)
• Two examples of consistent data

Helix A
21
Exceptions
?????
Met 374 (0.51 Å2) sulfur atom (0 Å2)
Met 385 (1.07 Å2) sulfur atom (0 Å2)
22
Molecular dynamic simulations
Time step 2 femptoseconds 3400 frames 6.8
nanosecond MD simulations
23
ASA for Met 374 during a 6.8 nanosecond MD
simulation
Met 374 Average 0.13 0.37 Å2 Max during
simulation 4.2 Å2 Calculated from crystal
structure 0.51 Å2
Sulfur atom of Met 374 Average 0.01 0.10
Å2 Max during simulation 1.6 Å2 Calculated from
crystal structure 0 Å2
24
The transient exposure of Met 385 over the course
of 16 picoseconds during which it goes from
totally buried to exposing 7 square angstroms
and back to buried.
25
6. Comparison of footprinting and H/D exchange
Consistent data
A
B

• unoxidized slow H/D exchange rate
• Helix B
• stand 1b, 2b, 3a and N-terminus of strand 3C
• oxidized fast H/D exchange rate
• RCL
• Helix F
• Strand 6a

Back
Front
D
C
Red oxidized yellow Unoxidized
26
Pre-existing flexibility in the F-helix
observed by both MS methods
27
7. Comparison of footprinting and H/D exchange
conflicting data
H/D footprinting Why
Strand 4c
H 209 slow oxidized
P 326 slow oxidized
M 374 slow oxidized ?
M 385 slow oxidized ?
P 255 slow oxidized ?
Strand 2C high unoxidized
no exposed reactive residue 14 fast/24 stand
6A helix H
Different capabilities of each MS method
28
Comparison of footprinting and H/D exchange
conflicting data

• Native a1AT are suboptimally folded
• 23 cavities, volume of 850 Å3
• Cavities side chain overpacking
• Cavity-filling mutations
• Cavity size nature of newly introduced residue
• Stabilizing effect size, shape of cavities
  flexibility of their local environment

29
Summary

• Compare the complementarity of two structural MS
  methods.
• H/D exchange better coverage/characterize
  the distribution of conformational flexibility
• Footprinting higher resolution/ reveal
  dynamic fluctuation of side chain
• Highlight differences between the static crystal
  structure and the dynamic conformation of ?1AT in
  solution reveal the structural designs that are
  critical for the biological function of ?1AT.

30

• Synchrotron footprinting characterization of
• ?1-AT/protease complex structure

Gupta S. et al., J. Synchrotron Rad. (2007) 14,
233-243
31
Serpin structures I

• Uncomplexed serpins
• First structure of a serpin was obtained in 1984
  (cleaved human ?1-PI)
• 1994, the first structures of uncleaved forms of
  inhibitory serpins were reported
• 30 additional structures for a total of 12
  different uncomplexed serpins have been reported
• Serpin/protease complex structures
• 2000, ?1-AT/trypsin
• Structure of a serpin-protease complex shows
  inhibition by deformation, Nature,
• Vol 407 923-926.
• 2006, ?1-PI/procine pancreatic elastase (PPE)
• Active site distortion is sufficient for
  proteinase inhibition by serpins, JBC, Vol 281,
• 3452-3457.

The extreme proteolytic susceptibility of the
complex coupled with the high concentrations
required for protein crystallization result in a
level of heterogeneity incompatible with crystal
growth.
?
32
Serpin structures II
33
?1-PI/PPE complex structures
Yellow free PPE Green PPE in complex
?1-AT/trypsin complex may have been
self-proteolyzed!
Active site distortion is sufficient for
proteinase inhibition by serpins, JBC, Vol 281,
3452-3457.
34
Synchrotron footprinting characterization of
?1-AT/protease complex structure
Strategy I
Forming a1 AT/trypsin complex (11)
Activity assay of free a1 antitrypsin
Calculate ? complex
Chromatography
Purified complex will be exposed to X-ray beamline
Digestion
LC-MS
35
?1AT/trypsin formation and purification
36
Trypsin gtgt HNE PPE 30 mins, pH 8.0, room
temperature are good, free AT is a C-terminal
truncated form.
10 µM AT incubated with 10µM porcine pancreatic
elastase (PPE) in potassium phosphate buffer, pH
8.0, room temperature
3hr
30 min
AT
complex
37kDa
truncated AT
PPE
PPE
11 1.5 1 1 1.5 11 1.5 1
1 1.5
Ratio is protease AT
37
Synchrotron footprinting characterization of
?1-AT/HNE complex structure
No enough Complex! No good separation!
T pH
Forming ?1AT/trypsin complex (11)
Free AT always exist!

• mg level

2) Trypsin 50mg for 25 HNE 5 mg for
15,100 PPE 5 mg for 151
38
Materials and Methods
Forming complex
Ratio 1.51 (AT/protease) Protein concentration
10 uM Incubation time 30 minutes Buffer 10 mM
phosphate buffer, pH 6.5 for purification 10 mM
phosphate buffer, pH 8.0 for incubation T Room
temperature
Exposed to X-ray
Run SDS-PAGE
complex
66kDa
a1 AT
44kDa
elastase
22kDa
Exposure
Cut three bands
Add Met.NH2 Add protease inhibitor
In gel digestion
LC-MS
-80 degree