Technique for probing protein structure and its conformational dynamics

1. K.LOHITHA
PA/2016/106
HYDROGEN
DEUTERIUM
EXCHANGE MASS
SPECTROMETRY
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2. PRESENTATION
FLOW
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3.  H/D-Exchange-MS has emerged as an important
technique to probe protein structure and conformational
dynamics
 H/D-Exchange-MS is a powerful approach for mapping
Protein folding
Protein-ligand interactions
Protein-protein interactions and
Conformational changes
 Companies have started using HDX-MS data as higher
order structure characterization of protein therapeutics
for regulatory agency filing.
INTRODUCTION
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4.  H/D-Exchange experiments are based on the chemical
reaction of replacing covalently bonded hydrogens with
deuterium atoms to reveal the tertiary structure
information of proteins.
 A peptide contains a large number of labile hydrogens
(O-H, N-H and S-H) that exchange with those from
surrounding water molecules.
 To monitor the exchange of amide hydrogens with the
solvent, proteins are diluted into an excess of deuterium
oxide (D2O).
 After exchange, the proteins are separated, ionized and
analysed.
THEOR
Y
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5.  A typical HDX mass spectrometry consist of 4 main
steps :
METHODOLOGY
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6.  When a protein solution (100-250µM) is
diluted 20-fold in D2O buffer
(99.5%pure) at 25°C & pH 7.0, labile
hydrogen atoms on the molecule will
exchange with deuterium.
 The rate of exchange between
backbone amide hydrogens and bulk
solvent depends on :
→ pH
→ Temperature
→ Hydrogen bonding
→ Solvent accessibility and
→ Dynamics
HYDROGEN-DEUTERIUM
EXCHANGE
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7.  There are 3 kinds of hydrogens in proteins.
 Hydrogens covalently bonded to carbon – do not exchange
 Side chain hydrogens - exchanges very fast & cant be detected
 Backbone amide H’s -
 Therefore, exchange rates are a reflection of structure and structural
stability
• Exchange at rates that can be measured
• Involved in formation of H-bonds with 2°
structural elements - both αhelices & βsheets
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8.  High solvent accessibility hydrogens exchange
quickly
(within µsec-msec)
 Presence of Hydrogen bonding hydrogens do not
(i.e., have a high protection factor) exchange quickly
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9.  This labeling (H/D-Exchange) is allowed to proceed for
defined periods of time (e.g., 10s, 1min,1h, 8h).
 Deuteration is not a one-way street: molecules in solution
will exchange back and forth dynamically, so in order to
measure the process accurately, the exchange had to be
chemically quenched to a pH of 2.5 (for proteins) and the
analytical separation simultaneously cooled to 0 °C to
manage the "back" exchange.
 Under quench conditions, the half-life for exchange in
unfolded polypeptides is ~1hr (meaning that if deuterated
protein were suddenly exposed to a 100% H2O
environment, it would take ~1hr for half of the deuterium
to exchange back to hydrogen).
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10. pH 5.0
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11.  After quenching, digestion is intended to break the
protein into peptides so that the location of deuteration
can be determined which increases spatial resolution
 Pepsin is preferred as the acid protease because of its
maximal activity at low pH.
 Although pepsin cleaves a protein randomly (unlike
trypsin or chymotrypsin) the digestion pattern is
consistent at constant temp & p/z ratio.
PROTEIN
DIGESTION
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12.  The generated peptides are loaded onto a C-18 reverse
phase HPLC column (0°C).
 The peptides are separated and eluted using an
acetonitrile gradient (0-60%) and analyzed for mass to
charge ratio (m/z) using a mass spectrometer.
SEPARATION
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13. IONIZATION :
The protein sample is subjected to electrospray ionization
The charged droplets are desolvated causing
accumulation of charge.
The electrostatic repulsions between the positively
charged particles cause them to fragment into smaller
droplets.
The process is continued until a fine spray of aerosol is
formed. The electrostatic potential help direct the positively
charged particles into the mass spectrometer
MASS
SPECTROMETRY
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14.  The peptides are separated according to the m/z and
the most intense ions (usually 4-10) are subjected to
collision induced dissociation using a neutral gas
(helium or argon).
 This collision further fragments the chosen peptide ions
into smaller ions which are again analyzed for m/z for
the mass spectrometer
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15. MASS ANALYZERS :
 Magnetic sector: uses a magnetic field to separate
 Quadrupole: uses a combination of RF fields and voltage
to separate
 Ion trap: a 3D quadrupole, uses RF and electric fields to
separate
 Time-of-flight: separates with time. Heavier molecules
take longer to fly down a tube than lighter molecules
DETECTOR:
Electron multipliers
Diode array detectors
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16. Why using HDX-
MS ?
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17. ADVANTAGES OVERNMR& X-
RAY
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18. 05/01/17
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19.  HDX-MS is a widely applicable straight forward
technique.
 Requires miniscule amount of protein (500-
1000picomoles for entire experiment including analysis
of 10 time points of exchange.
 Concentration of protein can be as low as 0.1µM
 Even large protein complexes can be studied.
 Membrane proteins, nearly impossible with other
techniques, can be studied.
ADVANTAGES
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20.  Analysis of HDX-MS data can be time-consuming
 Automation of the labeling and automation of the data
processing
 Above all, HX-MS needs to become a “turnkey”method
with ultrafast turnaround in an integrated platform
 Major limitation in terms of chromatography – separation
must be done at 0°C where chromatographic efficiency in
traditional HPLC is relatively poor
HDXworkbench
HXexpress
Mass spec studio
Hexicon2
DynamX
UPLC & new
separation media
LIMITATIONS
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21. APPLICATIONS
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22.  Two populations in mass
spectra
1 – folded state (blue
distribution)
2 – unfolded state (red
distribution)
 Appearance of 2
distributions occur if
Rate of interconversion
<amide
(folded & unfolded) exchange
 If a molecule unfolds –
becomes totally deuterated –
higher mass
FOLDING &
UNFOLDING
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25. 05/01/17
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26. CASE STUDY
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27. This article de als with comparison of the mAb samples
(same product) from three different manufacturing
processes (A,B,C)
Equipment
 Nano-UHPLC system with waters HD
technology
 Q-TOF mass spectrometer
 Data analysis software – DynamX
 Deuterium uptake levels over a time span of 10 s to 4
h were obtained for all the 3 intact mAbs
 A positive control experiment was conducted to
compare the deuterium uptake between a mAb
sample treated with 1.5M Guanidine-Hcl for 1hr with
the same mAb untreated. 05/01/17
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28. RESULTS &
DISCUSSION
Average RSD for individual
labeling time points was less than
5% (high reproducibility of the
methodology)
No statistically significant
differences in measured HDX
levels were observed at any
labeling time (indicating that there
was no global conformational
difference among 3lots of mAb )
Deuterium uptake for the
treated sample was higher
than the untreated sample
(sensitivity of the method is
high enough to differentiate
such global conformational
changes)
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29. CONCLUSION
 Hydrogen deuterium exchange mass
spectrometry is a sensitive technique to study
protein structure and dynamics in solution using
significantly lower protein concentrations than
crystallography and NMR spectroscopy.
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30. 2. Engen, J.R., Smith, D.L : Investigating protein
structure and dynamics by hydrogen
exchange MS. Anal. Chem. 73, 256A-265A (2001)
3. Wales, T.E., Engen, J.R : Hydrogen exchange mass
spectrometry for the analysis of protein dynamics.
Mass Spectrom. Rev. 25, 158-170 (2006)
4. Miranker, A., Robinson, C.V., Radford, S.E.,
Dobson, C.M : Investigation of protein folding by
mass spectrometry. FASEB J. 10, 93-101 (1996)
5. Skoog, D. A., Holler, F. J., & Crouch, S. R.
(2007).Principles of Instrumental Analysis.
1. Katta, V., Chait, B.T : Conformational changes in
proteins probed by hydrogen-exchange
electrospray-ionization mass spectrometry. Rapid
Commun. Mass Spectrom. 5, 214-217 (1991)
REFERENC
ES
6. Chalmers, M.J., Busby, S.A., Pascal, B.D., He, Y.,
Hendrickson, C.L., Marshall, A.G., Griffin, P.R :
Probing protein ligand interactions by automated
hydrogen/deuterium exchange mass spectrometry.
Anal. Chem. 78, 1005-1014 (2006)
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31. --- Brian Herbert ---
The capacity to learn is a gift;
The ability to learn is a skill;
The willingness to learn is choice.
--- Brian Herbert ---
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32. 05/01/1732