Rapid Mapping of Protein Conformational Landscapes by Automated
Data Acquisition & Analysis of Multiplexed HXMS Timecourses
Allis S. Chiena, Judith Frydmanb, Andrew D. Mirankerc, Sheila S. Jaswalb aVincent Coates Foundation Mass Spectrometry Laboratory,
bDept. of Biological Sciences, Stanford University, Stanford, CA; cDept. of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT; USA
Overview Methods Results STEP 4. Extract parameters from deconvoluted MS data,
Automated simultaneous monitoring of multiple HX An Advion NanoMate chip-based nanoelectrospray combine with known kint to calculate kclose.
reactions by TOF-MS & automated data analysis enable robot enables direct monitoring of aqueous protein STEP 2. Automated HXMS monitoring. a) Aliquots of
From amino acid sequence, aa seq ± pdb
multiplexed H/D exchange reactions and corresponding
the rapid characterization of protein folding behavior. solutions over a wide pH range, as well as reaction protonated controls are analyzed by nanoESI-MS; a NanoMate
temperature, pH: kint ≈ 60 s-1 ELVIS
temperature control (5-50°C). MS data is collected
Introduction using a Micromass Q-Tof mass spec operated in TOF
nanospray robot monitors reactions on pre-programmed
timecourses. b) Typical TIC and MS. Reaction start times are
Protein design carefully balances the disparate needs of mode; source pressure 6 mbar, cone V 180, source
staggered to interleave timepoints from the different reactions. From MS data:
c) Timecourse data at pH 7 and 8.5: protonated control and kex = 0.002 s-1
function, folding, and stability. Many proteins have temperature 30°C. kop = 0.0018s-1
HXMS spectra for +11 charge state of actin. m/z
native states to which they can spontaneously fold, but Deconvoluted experimental data
β2 microglobulin, 20% ACN, 32°C
they are also vulnerable to misfolding and aggregation, SCHEME 1. Overview of automated HXMS method
a) Automated reaction monitoring
Plug in values
leading to amyloid fiber formation in neurodegenerative Robot picks up pipet tip,
to equation for kop + kcl + kint − (kop + kcl + kint ) 2 − 4 • kop • kint kcl ≈ 8 s-1
2-state model: k ex =
aspirates reaction aliquot
and other amyloid diseases such as Parkinson’s. This 1) Set up H/D exchange reactions to cover EXX-EX2 range 2
mass spec source
phenomenon underscores the fact that the native state is
actually an ensemble of conformations, including rare 2) Monitor programmed timecourse with nanospray robot & mass spec
HX nonHX STEPS 5-6. Test models. 5) Simulate HX with extracted
partially and fully unfolded species, which can contribute rxns stds 6+ parameters; convolute parameters with protonated control
to the protein’s role in both biology and disease. Thus, denaturant spectra. 6) Global minimization: compare resulting
Mandrel rotates to
deliver sample through modeled data with experimental data; optimize to best match
understanding that role requires the ability to determine
Tx pHy m/z
nanospray nozzles in model & experimental.
the conformations populated from the native state, chip to mass spec
5) Test model with simulated HX
together with their stabilities and the kinetic barriers 3) Deconvolute raw data
b) Typical TIC & MS: 4 uM actin, 5 mM ammonium acetate, 37°C 0.002 s-1 if HX occurs through global unfolding (2-state,
using MS of protonated standard 60 s-1
between them under the physiological and pathological +12 +11
+ 8 s-1 no intermediate form)
conditions of interest. +
Simulate 2 state HXMS with extracted
# HX aa seq ± pdb ++
4) Extract parameters kop•kint + ELVIS +10 parameters, convolute with standard spectrum:
FIGURE 1. H/D exchange (HX) enables mapping of from deconvoluted data,
calculate unknowns kint,1-n Repeat steps 4-5 with other models (e.g. 3-state)
protein conformational landscapes. At equilibrium, native kop
known kint, 1-n calculate kcl Timecourse total ion chromatogram: MS: native folded protein,
each “block” is one time point few charge states
H/D exchange samples the entire landscape; degree of protection 6) Global minimization to find model & parameters which yield
from H/D exchange distinguishes native (N), intermediate (I) & 5) Model HXMS behavior: c) Parallel timecourses: MS of actin +11 charge state. Surface exposed best fit between experimental & simulated data
unfolded (U) conformations.1-5 Simulate HX distribution with kopen, kclose, kintrinsic,1-n protons exchange before 1st timepoint; protected protons exchange as
& convolute with pre-HX spectrum protein unfolds. Protonated control is shown in orange and simulated fully
CO deuterated in red (at end) for reference.
Ca CO Ca
protonated Ca N Ca N 3860
3880 3900 3920 3860
3880 3900 3920
Progress has been made toward developing a widely
protein 0 0
6) Compare modeled & observed, global minimization
deuterated H kint D for best fit of parameters to determine
pH 7 pH 8.5
applicable, general method for mapping protein folding
Accessible amide protons exchange with a. Is there an intermediate? or
landscapes via HXMS. Future work involves
deuterated solvent at known intrinsic rate (kint) b. What are unfolding & folding barriers & relative stabilities?
Funnel Landscape view c. What is slow exchange core in native form and degree of kcl kop comparative studies on analogous chaperonin-
U kopen kint kclose structure in intermediate form(s)? I K N 40 40
dependent and independent systems as well as further
Closed Open Exchange Closed
stable kclose streamlining and automation of the method.
Software tools assist in planning experiments by
y 20 0
STEP 1. HXMS reaction setup. a) A series of related H/D 10
simulating expected behavior for a given protein amino
I2 exchange reactions which cover the EXX-EX2 range are set up. acid sequence, identifying ranges of conditions (pH,
more N protons buried in folded proteins b) Practical considerations: evaporation of the reaction solutions temperature, denaturant) under which unfolding and
exchange through unfolding events over long reaction times (up to 72 hours) is prevented by addition
configuration STEP 3. Deconvolute raw MS data. Peak shape is folding rates are accessible.
of a hydrocarbon layer on top of the aqueous reaction.
broadened by deuterium uptake. Deconvolution subtracts the Additional software will automate the data deconvolution
kHX = kop kHX = (kop/kcl)• kint complex pattern of the mixture of actin species in the protonated
FIGURE 2. ESI-MS monitoring of H/D exchange. Observed and analysis described in steps 3-6.
larger ∆G,slower k
larger ∆G,slower k
a) Manipulate between regimes control from the H/D exchange data.
MS behavior of protein indicates kinetic regime of exchange. • slower kcl: increase temperature The software tools developed in this project will be made
and/or amount of denaturant
U Exch a) Raw MS of chosen b) Mathematical publicly available; information & links available through
Exch • faster kint: increase pH charge state of transformation
6+ N U N
protonated control to single peak
Unfolding Barrier (EX1) Stability (EX2) References
kint / kcl ≥ 10 kint ≈ 10 sec-1 kint / kcl ≤ 0.1 A; Nielsen, SO (1966). Adv Prot Chem 21, 287-386.
m*/z (m + a) /z (m + b) /z (m + c) /z
*after immediate HX of surface protons m/z kcl: 0.5 – 1 sec-1 at 25°C, pH 7 kcl: 50 – 100 sec-1 2 Woodward, C.; Carulla, N.; Barany, G. (2004). Meth Enzymol 380, 379-400.
t=0 t = end 3Bai,
b) Liquid evaporation barrier d) deconvolute c with the same Y; Sosnick, TR; Mayne, L. Englander, SW (1995). Science 269, 192-7.
What happens in between? c) Raw HXMS data 4Chamberlain, AK; Handel, TM; Marqusee, S. (1996). Nat Struct Biol 3, 782-7.
• prevents evaporation which would change protein concentration & kinetics function used to transform a to b 5Mayo, SL; Baldwin, RL. (1993). Science 262, 873-6.
(kopen /kclose)•kint • enables reactions at high temperatures (up to 50°C) 60 60
• compatible with robotics: pipet tip samples reaction through hydrocarbon layer
Density Vapor 0 0 Special thanks to Gary Schultz, Advion Biosciences; Mike Daly,
(g/mL, BP pressure
25°C) (°C) (mm Hg) Waters Corp.; the Vincent and Stella Coates Foundation; Stanford
Decane (C10) 0.73 174 1 (16.5°C) University Beckman Center Senior Research Fellowship to SSJ.
m/z m/z Dodecane (C12) 0.75 215-217 1 (47.8°C) This poster may be downloaded from the Stanford University Mass
population shift, kint >> kcl mass shift, kint ≈ kcl Simulation: aqueous solution & dodecane layer
Spectrometry website at http://mass-spec.stanford.edu/Publications.html