This document discusses hydrogen-deuterium exchange mass spectrometry (HDX-MS). It begins with an introduction that explains how HDX works by replacing protein hydrogens with deuteriums. It then covers the methodology which involves diluting proteins in deuterium oxide, followed by digestion, separation via liquid chromatography, ionization via electrospray, and mass analysis. The document discusses the theory behind HDX, noting regions of higher solvent accessibility exchange faster. It provides examples of applications like mapping protein folding and interactions. Finally, it presents a case study comparing three antibody samples and concludes that HDX-MS is a sensitive technique for studying protein structure and 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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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