Conformer-specific characterization of nonnative
protein states using hydrogen exchange and
top-down mass spectrometry
Guanbo Wang, Rinat R. Abzalimov, Cedric E. Bobst, and Igor A. Kaltashov1
Department of Chemistry, University of Massachusetts–Amherst, Amherst, MA 01003
Characterization of structure and dynamics of nonnative protein
states is important for understanding molecular mechanisms of
processes as diverse as folding, binding, aggregation, and enzyme
catalysis to name just a few; however, selectively probing local
minima within rugged energy landscapes remains a problem. Mass
spectrometry (MS) coupled with hydrogen/deuterium exchange
(HDX) offers a unique advantage of being able to make a distinction among multiple protein conformers that coexist in solution;
however, detailed structural interrogation of such states previously remained out of reach of HDX MS. In this work, we
exploited the aforementioned unique feature of HDX MS in
combination with the ability of MS to isolate narrow populations
of protein ions to characterize individual protein conformers
coexisting in solution in equilibrium. Subsequent fragmentation
of the protein ions using electron-capture dissociation allowed us
to allocate the deuterium distribution along the protein backbone,
yielding a backbone-amide protection map for the selected conformer unaffected by contributions from other protein states present in solution. The method was tested with the small regulatory
protein ubiquitin (Ub), which is known to form nonnative intermediate states under a variety of mildly denaturing conditions. Protection maps of these intermediate states obtained at residuelevel resolution provide clear evidence that they are very similar
to the so-called A-state of Ub that is formed in solutions with low
pH and high alcohol. Method validation was carried out by comparing the backbone-amide protection map of native Ub with
those deduced from high-resolution NMR measurements.
|
protein conformation protein dynamics
conformational dynamics
| nonnative state |
conformers might be segregated during the crystal growth stage
and characterized individually (11). Dynamic features of proteins
can be captured with NMR-based techniques (12), which, in
some cases, allow atomic-level characterization of nonnative
states to be achieved (especially in situations when nonnative
states can be stabilized by mutations or other alterations of protein
primary structure) (13–15). However, coexistence of multiple
conformers in solution still presents a challenge for NMR measurements, because the signals are typically averaged over the entire ensemble of protein molecules.
Hydrogen/deuterium exchange with mass spectrometry detection (HDX MS) is another powerful tool for characterizing
protein conformation and dynamics (16). One of the unique
features of HDX MS is its ability to detect and monitor individual protein conformers that become populated either
transiently under native conditions (17) or exist at equilibrium
under mildly denaturing conditions (18). Although such measurements can only be carried out under conditions that favor
the so-called correlated exchange (19), they are nonetheless extremely useful for characterizing complex energy landscapes
(20). The distinction among multiple protein states under these
conditions is made based on their different levels of deuterium
incorporation, which usually gives rise to bi- or multimodal isotopic distributions of protein ions in MS. Unfortunately, the
classical approach to characterizing protein structure with HDX
[i.e., proteolysis under the slow-exchange conditions, followed
by chromatographic separation of peptide fragments and MS
measurements of their deuterium content (21)] does not allow
separate characterization of individual conformers, because the
proteolytic fragmentation step is applied to the entire ensemble
of protein ions in solution regardless of their deuterium content.
T
he concept of protein energy landscapes, initially developed
to rationalize the fast acquisition of a unique native fold by
a fully unstructured polypeptide chain using the notion of a
“folding funnel” (1, 2), has been successfully applied in the past
decade to describe processes as diverse as recognition and
binding (3, 4), aggregation (5), enzyme catalysis (6), and other
functional properties (7, 8). The complexity of an energy landscape is usually emphasized by the presence of multiple local
minima, which frequently serve as important modulators of
protein function and other properties. Detailed characterization
of the structure and dynamics of these higher-energy (nonnative)
protein states is key to understanding the molecular mechanisms
of folding and other processes relevant for protein function
or homeostasis (9). However, selective characterization of individual nonnative states within a rugged energy landscape remains a formidable problem. Although spectroscopic techniques
frequently allow the presence of multiple conformers to be detected, they rarely provide the level of structural detail adequate
for complete characterization of these elusive species. Whereas
atomic-level structural details can be provided for native conformers by X-ray crystallography, characterization of multiple
states coexisting at equilibrium remains a challenge, although
some dynamic events can be visualized by trapping unstable states
(10), and, at least in some cases, highly structured near-native
www.pnas.org/cgi/doi/10.1073/pnas.1315029110
Significance
Structure and dynamic features of nonnative protein conformations are critically important for a variety of processes,
such as folding, recognition, binding, aggregation, and enzyme
catalysis to name just a few. Nevertheless, detailed structural
characterization of these elusive species is difficult, because
they almost always coexist in equilibrium with other conformers and cannot be isolated prior to analysis. As a result,
most studies report structural features that are averaged
across the entire protein ensemble, rather than unique features
of individual conformers. This paper reports structural characterization of an individual nonnative protein conformer without interference from other states coexisting in solution
under equilibrium.
Author contributions: G.W., R.R.A., and I.A.K. designed research; G.W. and R.R.A. performed research; C.E.B. contributed new reagents/analytic tools; G.W. analyzed data; and
G.W., C.E.B., and I.A.K. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1
To whom correspondence should be addressed. E-mail: kaltashov@chem.umass.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1315029110/-/DCSupplemental.
PNAS | December 10, 2013 | vol. 110 | no. 50 | 20087–20092
BIOPHYSICS AND
COMPUTATIONAL BIOLOGY
Edited* by S. Walter Englander, The University of Pennsylvania, Philadelphia, PA, and approved October 25, 2013 (received for review August 8, 2013)
Structural characterization of individual conformers might be
possible with the so-called “top-down” approach to HDX MS,
where the proteolytic fragmentation step in solution is replaced
with fragmentation of intact protein ions in the gas phase (22).
This approach has been used by several groups to probe protein
dynamics, although in all studies, the entire protein ion ensemble
was subjected to fragmentation to maximize the signal-to-noise
ratio, without selecting specific conformers (23–26). Top-down
HDX MS has been used to capture structural differences between
monomeric and oligomeric forms of a small amyloidogenic peptide (27, 28), as well as map the influence of posttranslational
modifications on protein higher order structure (29). Nevertheless, the ability of top-down HDX MS to probe structural and
dynamic features of individual monomeric conformers coexisting
in solution at equilibrium (by mass-selecting protein ions with
markedly different levels of deuterium incorporation before their
fragmentation) has never been demonstrated.
In this work, we used ubiquitin (Ub) as a model to demonstrate the unique ability of top-down HDX MS to extract
structural data on individual nonnative protein states without
interference from other conformers coexisting in solution at
equilibrium. Ub populates a partially structured state (the socalled A-state) in the presence of alcohol in acidic solution at
low salt (30), and this state was postulated to exist under a variety of other conditions (31), including those that favor correlated exchange regimes, where a distinction between this conformer and the native protein can be made based on markedly
different levels of deuterium incorporation in HDX MS measurements (32). The A-state of Ub, the structure of which has
been extensively characterized using NMR (33–38) and other
spectroscopic techniques (39), largely maintains the native secondary structure within the N-terminal part, whereas significant
conformational changes within the C-terminal part eliminate
the native fold and transform it into a transient helix. Top-down
HDX MS characterization of the nonnative conformer of Ub
reported in this work generates backbone-amide protection
patterns fully consistent with the A-state of the protein and
demonstrates that that top-down HDX MS/MS is capable of
selective characterization of structural features of nonnative
protein states without interference from other conformers
coexisting in solution at equilibrium.
Results
Method Validation: Top-Down HDX MS/MS Characterization of Native
Conformation of Ub and Comparison with High-Resolution NMR. The
single factor with the highest potential to compromise the quality
of HDX MS/MS measurements is hydrogen scrambling [i.e.,
intramolecular migration of labile hydrogen atoms causing
rearrangement of protons and deuterons within the protein ion
before its dissociation (40, 41)]. Earlier studies demonstrated
that electron-based fragmentation techniques result in negligible
hydrogen scrambling within protein ions (24, 25); nevertheless,
caution always needs to be exercised to ensure the absence of
scrambling, which may be caused, among other things, by collisional heating of protein ions before their fragmentation. In this
work, we minimized collisional heating by selecting relatively
gentle conditions of ion production, selection, and accumulation
before their fragmentation with electron-capture dissociation
(ECD) (see Materials and Methods for specific details), with
b and y type of fragment ions serving as indicators of collisional
heating (42). Although the absence of b and y ions in the mass
spectra of ECD fragments provides a reasonable assurance that
collisional heating of protein ions is minimal, additional evaluation of the extent of hydrogen scrambling was required. This
was achieved by comparing the backbone protection map deduced from the top-down HDX MS measurements on native Ub
with the previously reported NMR data (35, 37, 43).
HDX of native Ub was performed by exposing 2H-labeled Ub
to the 1H-based exchange buffer at neutral pH, low salt (10 mM
NH4CO2H) solution for 161 s (see exchange condition I in Table
1), and [M+10H]10+ protein ions were mass-selected for frag20088 | www.pnas.org/cgi/doi/10.1073/pnas.1315029110
mentation using a 20 m/z units-wide isolation window. Overall,
33 c ions and 34 ions were observed in the ECD mass spectrum
(Fig. 1), the ionic signals of which were sufficiently high to be
used for calculating distribution of deuterium across the protein
backbone (i.e., producing backbone-amide protection maps).
These fragment ions allowed a single-residue resolution to be
achieved for 84% of the protein sequence, with additional 11%
of the sequence resolved at two-residue resolution (note that
there are three Pro residues in Ub, which do not produce c and z
type of fragments, nor do they bear reporter hydrogen atoms on
their amide groups). The fraction of deuterium retained at each
individual backbone amide, D(Ri), was calculated as described in
Materials and Methods, and the results are plotted in Fig. 2 as
a function of the residue number in Ub sequence Ri (all numeric
values are presented in Table S1). The full theoretical range of D
(Ri) values is from 0 [indicating the complete exchange (i.e., no
protection of backbone amide)] to 0.91 (indicating full retention
of the deuterium label). The latter value is determined using the
1
H/2H ratio in the final protein solution (it would have been 1.00
in the case of infinite dilution of the protein stock solution in
exchange buffer during initiation of the exchange reactions).
The backbone-amide protection map of native Ub (Fig. 2)
shows excellent agreement with the X-ray crystal structure of the
protein (44), because all regions displaying high protection align
with the highly structured segments of the protein (all elements
of Ub 2° structure are presented symbolically on top of the
protection map in Fig. 2). Additional evaluation of the quality of
the protection map was carried out by comparing it with three
sets of previously reported high-resolution NMR data (35, 37,
43). This was done using the procedure developed by Sterling
and Williams (45), where the exchange-rate constants determined by NMR are plotted on the normalized negative-logarithm scale (with 1.0 corresponding to the slowest rate and 0.0
to the fastest). Slight incongruence of the three datasets (represented with bars of different colors in Fig. 2) may be caused by
variations in experimental conditions, including solution pH,
buffer composition, ionic strength, and temperature. Overall, the
backbone-amide protection maps derived from the NMR
measurements are in excellent agreement with the top-down
HDX MS data obtained in this work. The very few residues
where significant deviation exists between the NMR and HDX
MS data (e.g., Ile36 and Ser65) also display noticeable discrepancy among different NMR sets. On the other hand, there is
a remarkable agreement between the NMR and HDX MS data
in the regions of alternating protection (e.g., β-strand 2, where
only every other amide hydrogen is involved in hydrogen bonding), suggesting that the top-down HDX MS correctly captures
structural features of protein conformation at high resolution.
Overall, comparison of the backbone protection pattern of native
Ub deduced from the top-down HDX MS data are in good
agreement with both X-ray crystal structure of this protein and
NMR measurements, suggesting that under the experimental
conditions chosen in this work the extent of hydrogen scrambling
is minimal and does not negatively affect the quality of HDX
MS data.
Isolation of Ionic Signals of Individual Conformers of Ub Coexisting at
Equilibrium. To interrogate structural features of a nonnative Ub
conformer coexisting at equilibrium with other protein states,
top-down MS measurements were carried out following HDX in
a solution with high alcohol content at neutral pH (see condition
II in Table 1). Ub is known to populate more than one conformation under these conditions (31, 32), one of which was previously hypothesized to be the classical A-state of Ub (the
commonly accepted way to generate the A-state of Ub uses an
acidic solution with high alcohol content). As illustrated in Fig. 3,
the isotopic distribution of intact Ub ions during HDX (Ub* in
Fig. 3A) clearly displays a bimodal shape under these conditions.
This bimodal character was previously postulated to reflect
transitions between the native state of Ub and a partially structured
nonnative intermediate occurring under conditions favoring the
Wang et al.
Table 1. Solution parameters for HDX reactions
Composition, % in vol/vol
Condition
Solution A
I
2
II
2
III
2
Solution B
2
H-labeled Ub in 10 mM NH4OAc, H2O
H-labeled Ub in 10 mM NH4OAc, 2H2O
H-labeled Ub in 10 mM NH4OAc, 2H2O
1
10 mM NH4OAc in H2O
10 mM NH4OAc
in 40% 1H2O and 60% CH3O1H
40% 1H2O, 60% CH3O1H
Solution C*
Room temperature, °C
50% H2O, 50% CH3CN,
formic acid
40% 1H2O, 60% CH3O1H,
formic acid
40% 1H2O, 60% CH3O1H,
formic acid
28 ± 1
1
24 ± 2
25 ± 2
*The requisite amount of formic acid was added to drop the pH of final mixture to 2.5.
1
21
41
61
M Q I F V K T L T G K T I T L E V E P S
D T I E N V K A K I Q D K E G I P P D Q
Q R L I F A G K Q L E D G R T L S D Y N
I Q K E S T L H L V L R L R G G
c ions
z ions
Fig. 1. ECD sequence coverage of Ub under conditions minimizing hydrogen scrambling.
Wang et al.
absolute signal intensity was diminished by only 31% and 33%,
respectively.
Structural Characterization of Individual Conformers. Mass comparison of ECD fragments derived from intact ions representing
isolated C-1 and C-2 conformers immediately highlights the
presence of protein segments where backbone protection is nearly
identical for the two conformers, as well as segments where the
protection difference is very significant (indicating significant difference between their structures). An example is shown in Fig. 4,
where the isotopic distributions of c142+ fragments (representing the
N-terminal segment of Ub containing the first 14 backbone amides)
derived from C-1 and C-2 conformers are virtually indistinguishable,
whereas the z142+ fragments (representing the C-terminal segment
of the protein containing thirteen backbone-amide groups) derived
from the same conformers exhibit noticeable difference. This simple
analysis suggests that the higher-order structures of C-1 and C-2 are
likely to be very similar in the N terminus of Ub, whereas significant
differences exist in its C terminus. It is also noteworthy that such
differences are not apparent when top-down HDX MS data are
acquired for the entire population of protein ions without isolation
of individual conformers (Fig. 4A).
The levels of retained deuterium label following exchange in
solution plotted for all fragment ions derived from isolated C-1
and C-2 conformers (Fig. 4D) also reveal a dramatic difference
between these two conformers in the C-terminal part of the
protein. To identify the regions exhibiting the most dramatic
structural differences, these data were used to calculate deuterium retention at individual amides, D(Ri), for both conformers
(Fig. 5 and Table S1). Protein residues exhibiting significant
difference in deuterium retention between C-1 and C-2 were
identified using the following criterion. The absolute value of the
most negative D(Ri) (0.15) (on the graph in Fig. 5) was treated as
the extreme magnitude of error oscillation [obviously, the theoretical, or error-free value of D(Ri) should never fall below 0].
Any residue Ri where the value of DC−2 (Ri) − DC−1 (Ri)
exceeded 0.30 (the double of 0.15) was regarded as a residue
with “significant difference” in protection between the two
conformers (highlighted in gray in Fig. 5).
Overall, there is a remarkable similarity between the backbone-amide protection maps of C-1 and C-2 within the N-terminal half of the protein (residues 1–35), whereas significant
differences are observed within the C-terminal half. Furthermore, the protection pattern of the C-2 conformer is remarkably
close to that of the native Ub (Fig. 2), consistent with the previously made suggestion (31, 32) that the structured state populated by the protein under these conditions (neutral pH, high
alcohol content) is in fact the native conformer (see SI Text and
Fig. S1 for more detailed analysis). To prove that the less structured conformer (C-1) is similar to the A-state of Ub, a reference
protection map was obtained by carrying out the exchange reactions in zero-salt solution containing 60% alcohol (vol/vol) (see
condition III in Table 1), which was previously shown to populate
the A-state of the protein (32). A detailed comparison of the two
protection maps (C-1 and A-state) reveals their near complete
PNAS | December 10, 2013 | vol. 110 | no. 50 | 20089
BIOPHYSICS AND
COMPUTATIONAL BIOLOGY
EX1 exchange regime, whereas transitions from either of these two
states to the unfolded (fully unstructured) state of Ub would occur
under conditions favoring the EX2 regime (32). This results in
a slow loss of deuterium label by both ion populations in the bimodal distribution shown in Fig. 3 without the explicit appearance
of ions representing the fully unstructured state (such as observed
for the endpoint sample in Fig. 3A). The two protein states clearly
visible in the isotopic distribution of ions Ub* in Fig. 3A were
denoted C-1 (the less protected conformer represented by ionic
signal at m/z 859–863) and C-2 (the more protected conformer
represented by ionic signal at m/z 861–864). Because replacement
of labile deuterium with hydrogen atoms in the course of HDX is
essentially an irreversible process, increase of the exchange time
results in a progressive accumulation of C-1 signal, whereas C-2 is
diminished. In this work, the duration of exchange (161 s) was
chosen such that the ionic signals representing the two conformations are nearly equal to each other (Fig. 3B). It is important
to note that the apparatus developed in this work allows the ionic
populations representing distinct conformers to be readily isolated
at different exchange time points by varying the length of the exchange loop and/or manipulating the flow rates.
Isolation of ionic signals of each conformer was achieved by
mass-selecting a fraction of [M+10H]10+ ions in the front-end
quadrupole filter. The center and width of the isolation windows
were chosen to eliminate ionic signal of the neighboring conformer while minimizing signal loss of the conformer being selected. Partial overlap of the ionic signals representing C-1 and
C-2 made it difficult to isolate C-1 without interference from the
other conformer: the shoulder in the envelope of isolated isotopic peaks of C-1 indicates the presence of residual C-2 signal
(Fig. 3C); however, the relative abundance of this signal is less
than 6%. Likewise, the isolated ionic signal of C-2 (Fig. 3D)
might also contain small interference from C-1 in the form of
sodium ion adducts falling within the ion isolation window (formation of a Na+ adduct results in a mass increase of the protein ion
by 22 Da). However, this interference was estimated to be less than
7% of the total isolated signal based on the net abundance of alkali
metal ion adducts in mass spectra of Ub (e.g., Fig. 3B). The isolated
signals of C-1 and C-2 conformers are centered at m/z 860.7 and
862.4, respectively, deviating from the centroids of the isotopic
envelops representing these conformers in the isotopic distribution
of Ub* before isolation by only 0.4 and 0.2 m/z units, whereas the
-log (exchange rate constant)
(by NMR)
Bougault, et al
Pan and Briggs
Johnson, et al
0.8
1.0
MS data
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
5
10
15
20
25
30
35
40
45
50
55
residue number
congruence, providing strong evidence that the C-1 conformer
resembles the A-state of Ub.
Discussion
Understanding structural properties of activated states of proteins is critical for deciphering molecular mechanisms of a range
of biological processes, and yet these elusive species frequently
remain difficult or indeed impossible to isolate and characterize
because of their transient nature and/or presence of other protein states that may interfere with the measurements. MS offers
a unique advantage of being able to detect and track multiple
protein conformers in solution under a variety of conditions by
either monitoring charge state distributions of protein ions in
electrospray ionization (ESI) mass spectra (46) or by recording
HDX profiles under conditions favoring correlated exchange
(47); however, detailed structural characterization of these states
remained out of reach of MS-based methods of structural analysis. Although fragmentation of the entire ensemble of protein
molecules in solution by means of proteolysis or in the gas phase
using various ion activation techniques frequently generates
fragments with convoluted isotopic distributions, even the most
sophisticated deconvolution methods (48) cannot establish unequivocal correlation between these distributions and those of
the intact protein ions. The approach used in this work to
A
C1
selection
window
Ub10+
853
side chain
backbone
Ub*
10+
C2
selection
window
Ub**10+
endpoint
858
860
862
864
866
868
870
872
m/z
B
cation adduct
C
D
cation adduct
858
859
860
861
862
863
864
865
m/z
Fig. 3. Isotopic distributions of intact Ub ions (charge state +10) before (A)
and after (B–D) precursor ion isolation. A shows the extent of 2H retention on
partially exchanged Ub (Ub*) by overlaying its spectrum with spectra of unlabeled (Ub), completely exchanged Ub (endpoint), and fully deuterated
(Ub**) protein ions on a zoom-out scale. B shows broad-band isolation of Ub*
ions, and C and D illustrate isolation of Ub* ions representing conformers C-1
and C-2, respectively (the isolation windows are shown in A).
20090 | www.pnas.org/cgi/doi/10.1073/pnas.1315029110
60
65
70
deuterium retention
at individual residue (by MS)
1.0
Fig. 2. Comparison of relative protection patterns
determined by top-down HDX MS (line-graph; closed
symbols represent data obtained from c ions, and
open symbols are from z ions) and NMR [white (43),
gray (37), and light-gray (35) bars]. The error bars
show the SD (based on a set of three replicate
measurements). MS data at double-residue resolution are highlighted. Secondary structural elements
of native state Ub (Protein Data Bank ID code 1UBQ)
are shown schematically on top of the diagram.
overcome this problem relies on physical isolation of protein ions
representing specific individual conformers based on their deuterium content, followed by their fragmentation. Even in the
relatively unfavorable case of Ub considered in this work (where
the isotopic distributions corresponding to the A-state and the
native conformer partially overlap), it is possible to isolate each
conformer’s signal such that contribution of the other conformer
does not exceed 6–7%.
The ability to physically isolate protein ions representing different conformers coexisting at equilibrium in solution before
using ECD to analyze the distribution of deuterium atoms across
the protein backbone allows their structural characterization
to be carried out in a highly selective fashion with minimal interference from other states. Isolation and structural interrogation
of the two (partially) structured equilibrium states of Ub populated
at neutral pH, low-salt solution in the presence of alcohol generates backbone protection maps, which are consistent with the Astate of the protein and its native conformation. Importantly,
structural details of both conformers can be seen at high (residuelevel) resolution. For example, not only can one clearly see the loss
of stable structure in the C-terminal part of the protein upon
formation of the A-state but also much smaller elements lacking
backbone protection (and, therefore, the ability to retain deuterium label), such as β-turns in both A-state and the native conformer of Ub (e.g., see segments Thr7-Lys11 and Lys33-Asp39 in
Figs. 2 and 5). An even higher level of detail can be seen in the
protein segment Thr12-Glu18 (corresponding to β-strand 2),
which shows a very uneven protection pattern (with only every
other amide group exhibiting high level of deuterium retention),
consistent with the topology of hydrogen bonding between a terminal strand in the β-sheet and the neighboring strand (38). This
level of detail allows all equilibrium states of Ub interrogated in
this work to be unambiguously identified using direct comparison
of their high-resolution backbone protection maps with the
“reference” maps of the known Ub conformers.
It remains to be seen whether the top-down HDX MS approach presented in this work is capable of teasing out even finer
differences in higher-order structure among various protein
conformers, such as making a distinction between the native
state of Ub and its higher-energy state N2 [populated at high
pressure or produced as a result of mutations (14, 49)], which has
a nearly identical secondary structure to the native state but
a slightly altered tertiary fold. Although physical isolation of ions
corresponding to the N2 state from those representing the native
conformer by means of MS/MS alone can prove problematic
because of the expected similarity in HDX kinetics, utilization
of ion-mobility spectrometry (50) can address this problem by
allowing the two conformers to be resolved temporally based on
the difference in their shapes in the gas phase.
We have demonstrated the capability of top-down HDX MS
to specifically capture structural features of individual protein
conformers coexisting in solution with high spatial resolution.
Structural differences were evident in protection maps obtained
at a single exchange point for Ub under conditions known to
induce multiple conformations, and collecting HDX datasets at
various time points would permit the exploration of HDX kinetics, providing an additional dimension to structural characterization. The methodology described in this work is applicable
Wang et al.
relative abundance
ensemble
B
C-1
ΔM=2.9
C-2
D 782
cumulative
deuterium retention
ΔM=1.0
ΔM=2.3
C
784
20
786
788
790
792
794
796
906 908 m/z
c ions of C-1
z ions of C-1
c ions of C-2
z ions of C-2
c142+
15
z162+ z 2+
14
10
5
0
5
10
15
20
25
30
35
40
45
residue number
50
55
60
65
70
Fig. 4. Isotopic distribution of c142+ and z142+ fragment ions generated
upon ECD of intact Ub* ions representing either the entire ensemble of
protein molecules (A), the C-1 conformer (B), or the C-2 conformer (C ).
Vertical dashed lines represent the calculated centroids of the corresponding isotopic distributions, and line widths indicate the SDs. D shows
the cumulative deuterium retention within protein segments plotted as
a function of the segment’s length counting from either the N or C terminus of the protein (the dashed line separates the data obtained from
c and z ions).
to any intact protein, as long as it is possible to select conditions
when the intrinsic (chemical) exchange rate is significantly higher
than the refolding rate of the partially unfolded protein back to
its native state. Although this does rule out the possibility of
directly probing the structure of transient nonnative states under
the native conditions (which rarely favor the EX1 exchange regime), structural information obtained under the mildly denaturing conditions for the equilibrium nonnative states is still
very valuable for understanding the mechanisms of a variety of
processes where nonnative protein states play important functional roles, such as ligand recognition/binding (51) and protein
association (52, 53). The unique capability of the top-down HDX
MS demonstrated in this work coupled with other MS-based
methods of macromolecular structure analysis, such as monitoring charge-state distributions of protein ions and ion-mobility
spectrometry, is likely to open new possibilities in the fields of
biophysics and structural biology. Characterizing the structure of
partially folded (nonnative) states of proteins in a highly individualized fashion without interference from other conformers
that may either coexist at equilibrium or become populated
transiently will provide valuable information needed to further
our understanding of a plethora of biological processes involving
proteins and reveal the most intimate details of their behavior
relevant not only to function, but also misfolding, aggregation,
and amyloidosis.
Materials and Methods
Materials. Ub from bovine erythrocytes was purchased from Sigma-Aldrich.
Deuterium oxide (99.9% 2H) was purchased from Cambridge Isotope Laboratories. Ammonium acetate, acetic acid, and methanol (HPLC grade) were
purchased from Fisher Scientific.
stock solution of Ub** (solution A in Table 1) was kept at 4 °C. The extent of
deuteration of Ub** was verified by ESI MS, yielding a 97.4% replacement of
labile hydrogen atoms with deuterium. HDX was initiated by mixing Ub**
with 1H2O-based exchange solvent (solution B in Table 1) in an in-house,
online-mixing apparatus that comprises three inlets and two sequential
high-efficiency mixing sites regulated by Nano-Mixer chips (Upchurch Scientific), as depicted in Fig. S2. Three syringes used to infuse protein solution,
exchange solvent and quench solvent (solutions A, B, and C in Table 1) were
advanced simultaneously by a single Nexus 3000 syringe pump (Chemyx),
resulting in a constant 10-fold dilution of solution A into B at the first mixing
site, and acidification of resulting mixture with solution C at the second
mixing site. Whereas the exchange loop (between the two mixers) was kept
at ambient temperature, the quench solvent (solution C in Table 1) and all
capillaries downstream of the second mixer were kept on ice to minimize
possible loss of 2H label by the protein past the exchange loop. The length of
the capillary comprising the exchange loop was selected to allow the exchange reactions to proceed for 161 s at the selected flow rate (297 μL/h).
The setup was equilibrated for one hour before each measurement. The
endpoint sample was prepared by mixing solutions A, B, and C at the appropriate ratio followed by an incubation at 37 °C for 2 h before ESI MS.
ESI MS Measurements. ESI MS measurements were carried out with a SolariX 7
(Bruker Daltonics) Fourier transform ion cyclotron resonance (FT ICR) MS. The
ESI source was directly connected to the outlet of the online mixing apparatus. Mass-selection of [M+10H]10+ ions in the front-end quadrupole filter
was followed by ECD of precursor ions in the ICR cell. Typically, 3,000–4,000
scans were accumulated for each ECD spectrum. To ensure scrambling-free
fragmentation of protein ions, relatively mild ion desolvation and isolation
conditions were used in this work (capillary exit: 190.0 V; collision voltage: −3.0 V;
collision RF amplitude: 500.0 V. Isolation windows for Ub ions representing
conformers C-1 and C-2 are as follows: C-1, centered at m/z 857, width 7.5 m/z
units; C-2, centered at m/z 865.5, width 7.0 m/z units.
HDX MS/MS Data Analysis. Masses of fragment ions were determined as
follows: for those peak clusters whose monoisotopic peak was more intense
than the last peak used for calculation, number average masses were calculated; otherwise, for sake of reducing error, the isotopic envelope was fit to
a Gaussian curve after discarding any overlapping peaks to determine the m/z
of its apex (xC) (54), followed by mass determination using xC (see SI Text and
Fig. S3). The cumulative backbone-amide 2H retention of a certain segment
represented by c or z ions was calculated as:
DðSi ðNÞÞ =
DðSi ðCÞÞ =
M* ðci−1 Þ − Mðci−1 Þ
M 2H − M 1H
− NEX ðci−1 Þ × 0:97=11;
M* ðzi+1 Þ − Mðzi+1 Þ
M 2H − M 1H
− NEX ðzi+1 Þ × 0:97=11,
where Si(N) or Si(C) are segments extending from either the N or C terminus to the ith residue; M*(ci) or M*(zi) was the mass of deuterated ci or
N
A
A
Conformer C-1
1.2
Conformer C-2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
B
5
10
15
20
25
30
35
40
45
50
55
60
65
70
1.2
native state
A-state
1.0
0.8
0.6
0.4
0.2
0.0
Hydrogen Exchange. Ub was fully deuterated (Ub**) by repeated cycles of
incubation in deuterium oxide (2H2O) with 3% (vol/vol) formic acid for 90
min at 40 °C, followed by lyophilization; 2H2O was used to dissolve Ub
subsequent to the final round of lyophilization and then a 200 mM solution
of ammonium acetate made up with 2H2O was added to yield a final concentration of 10 mM ammonium acetate and 20 μM Ub; this HDX-ready
Wang et al.
-0.2
5
10
15
20
25
30
35
40
45
50
55
60
65
70
residue number
Fig. 5. Comparison of backbone protection maps for C-1 and C-2 (A) and
for the native and A-state of Ub (B). Closed and open symbols represent
data obtained from c and z ions, respectively. Secondary structural elements
of native and A-state Ub are depicted on top of the diagram.
PNAS | December 10, 2013 | vol. 110 | no. 50 | 20091
BIOPHYSICS AND
COMPUTATIONAL BIOLOGY
z162+
c142+
deuterium retention
at individual residue
z142+
deuterium retention
at individual residue
A
zi ions; NEX was the total number of exchangeable hydrogen atoms contained in the segment. Mass of unlabeled species M was obtained experimentally. Amide retention at an individual backbone-amide group was
determined as:
for 1 < i < 36, DðRi Þ = DðSi ðNÞÞ − DðSi−1 ðNÞÞ;
D Si+j ðNÞ − DðSi ðNÞÞ
;
for 1 < i < 36, DðRi Þ = DðRi+1 Þ = . . . = D Ri+j−1 =
j
D S77+j−i ðCÞ − DðS76−i ðCÞÞ
for 36 < i<76, DðRi Þ = DðRi+1 Þ = . . . = D Ri+j−1 =
:
j
For residues where subsequent fragment ions were missing, D(R i) was
assigned as:
ACKNOWLEDGMENTS. We thank Ms. Yao Lu (Regeneron, Inc.) for helpful
discussions. This work was supported by National Institutes of Health Grant
GM061666. The Fourier transform ion cyclotron resonance mass spectrometer was acquired with support from National Science Foundation Grant
CHE-0923329 (Major Research Instrumentation Program).
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for 36 < i<76,
DðRi Þ = DðS77−i ðCÞÞ − DðS76−i ðCÞÞ:
20092 | www.pnas.org/cgi/doi/10.1073/pnas.1315029110
Wang et al.