A Triaxial Probe for On-line Proteolysis
Coupled with Hydrogen/Deuterium
Exchange-Electrospray Mass Spectrometry
Maolian Chen and Kelsey D. Cook
Department of Chemistry, University of Tennessee, Knoxville, Tennessee, USA
Center for BioModular Multi-Scale Systems, Louisiana State University, Baton Rouge, Louisiana, USA
Graduate School of Medicine, University of Tennessee, Knoxville, Tennessee, USA
An on-line proteolysis system utilizing a triaxial electrospray probe was developed to aid
localization of the hydrogen-bonding interaction sites in hydrogen/deuterium exchange-mass
spectrometry (HDX-MS) studies of A? (1-40) fibrils. The probe allows delayed introduction of
the organic solvent component needed for stable electrospray, thus enhancing hydrolysis
performance relative to that of a coaxial probe. Effective on-line digestion was accomplished
in ?12 s. The probe should be of general utility for HDX-MS studies of amyloid fibrils and
other protein aggregates. (J Am Soc Mass Spectrom 2007, 18, 208–217) © 2007 American
Society for Mass Spectrometry
the fact that amide protons on the backbone of proteins
exchange with solvent protons or deuterons at rates that
depend on whether they are involved in hydrogen
bonds in secondary structural elements such as ?-
helices and ?-sheets, and/or whether they are sterically
shielded from the solvent. Analysis normally involves
exposing a protein or noncovalent complex to exchange
conditions in a D2O-based buffer solution, followed by
measuring the incorporation of deuterium using meth-
ods such as magnetic resonance [1–2], infrared spec-
trometry [3– 4], or mass spectrometry (MS) [5–7].
HDX-MS is well-suited to analysis of relatively large
and soluble proteins and to protein mixtures [5–7].
The measured mass shift upon deuteration in
HDX-MS analysis provides insight into the overall
exchange protection of the entire protein molecule.
Determination of specific exposed and protected sites
by HDX-MS requires coupling with proteolysis [6, 8]
and/or tandem mass spectrometry (MS/MS) , gen-
ydrogen/deuterium exchange (HDX) has been
widely used to probe structural and dynamic
features of proteins [1–7]. The method exploits
erally with collision-induced dissociation (CID) or
(more recently) electron capture dissociation (ECD)
. There is some risk of intramolecular scrambling of
deuterium labels during CID [11, 12] or ECD .
Peptide digestion can offer an alternative or supple-
ment to direct CID or ECD for localizing incorporated
deuterium with HDX-MS. Pepsin has proven to be
particularly useful for this purpose because of its good
activity at the low pH values used to minimize artifac-
tual hydrogen exchange (spurious incorporation or loss
of deuterium) during sample work-up after exchange.
A typical protocol involves incubation of deuterium-
labeled proteins with pepsin at about pH 2 for ?5 min
at 0 °C (low-temperature also reduces artifactual ex-
change). The digest is then subjected to liquid chroma-
tography (LC), with on-line MS or MS/MS analysis to
assess the deuterium content of individual digestion
products [3–5, 8]. Relatively high quantities of pepsin
are used to minimize digestion time and the corre-
sponding opportunity for scrambling; this can compli-
cate direct analysis of the hydrolysate without LC
separation (due to increased background) . Use of
an immobilized enzyme in a continuous flow column
can mitigate these problems [14 –16].
We have adapted electrospray HDX-MS methodolo-
gies for analysis of the A? amyloid fibrils associated
with Alzheimer’s disease [17–19]. To dissolve fibrils
after deuteration and before MS analysis while limiting
artifactual exchange, we have used continuous flow
mixing of the deuterated fibril suspension with a
Published online October 30, 2006
Address reprint requests to Dr. K. D. Cook, Department of Chemistry,
* Current address: Pennington Biomedical Research Center, Louisiana State
University System, Baton Rouge, LA, USA.
† Current address: Department of Structural Biology, Pittsburgh Institute
for Neurodegenerative Diseases, University of Pittsburgh School of Medi-
cine, Pittsburgh, PA, USA.
© 2007 American Society for Mass Spectrometry. Published by Elsevier Inc.
Received July 13, 2006
Revised September 17, 2006
Accepted September 18, 2006
low-pH dissolving solvent that contains acetonitrile
(MeCN) to facilitate electrospray and to reduce artifac-
tual exchange (by reducing the protic solvent concen-
tration in the final mix). Using this approach, we have
determined that roughly half of the 39 A? (1-40) amide
protons are protected from HDX in fibrils grown from
the peptide [17, 18]. In anticipation of efforts to further
localize the protected sites, we needed a way to achieve
rapid proteolysis with minimal scrambling. Use of an
immobilized enzyme would require fibril dissolution
before passing through an enzyme column; a faster
sample treatment protocol would be desirable. We
demonstrate here the utility of a triaxial electrospray
probe to facilitate effective and rapid on-line dissolution
and proteolysis of fibrils at room temperature. The
performance is compared with that of the coaxial probe
used previously [17, 18].
HPLC-grade acetonitrile (MeCN) and water were pur-
chased from Fisher Scientific (Pittsburgh, PA). Trifluoro-
acetic acid (TFA) was purchased from Pierce Biotechnol-
ogy Inc. (Rockford, IL). Deuterium oxide (100.0 atom %D)
was purchased from Aldrich Chemical Co. (Milwaukee,
acid (95% in water), tris(hydroxymethyl)aminomethane
(tris), and porcine pepsin were obtained from Sigma
Chemical (St. Louis, MO). Triethylamine was obtained
from Acros (Pittsburgh, PA) and was distilled before use.
All other reagents were used as received. Stock solutions
of pepsin were prepared daily at a concentration of 2
?g/?L in 0.01M HCl. Working solutions were prepared
of water, MeCN, and/or formic acid to generate solutions
with final composition consistent with the desired MS
processing solvent. Nitrogen gas for electrospray experi-
ments was obtained from liquid boil-off.
A? fibrils were grown in phosphate buffer from chem-
ically synthesized A? (1-40) peptide (Keck Biotechnol-
ogy Center, Yale University, New Haven, CT) accord-
ing to the protocol described previously . Fibril
growth was monitored by thioflavin T fluorescence and
the quality of fibrils was confirmed by electron micros-
copy. To remove the phosphate buffer before MS anal-
ysis, fibrils were collected by centrifugation (Eppendorf
Centrifuge 5415C, Brinkmann Instruments, Inc., West-
bury, NY) at 14,000 rpm for 30 min. After decanting and
discarding the supernatant buffer, the fibrils were
washed once with 2.0 mM tris·HCl or tris·DCl buffer
(pH 7.5), and then recentrifuged, decanted, and resus-
pended in fresh tris buffer. Monomer samples were
dissolved directly in tris buffer. Fibril and monomer
samples were prepared at equivalent monomer concen-
trations between 5 and 42 ?M, as noted.
To assess the effect of acetonitrile on pepsin activity,
A? monomer solution in 2 mM tris was digested with
freshly-made pepsin solutions in the presence of water,
0.5% formic acid, and varying amounts of MeCN (0, 15,
30, 50% vol/vol). Solutions also contained 0 or 0.06
mg/ml (?14 ?M) A? monomer and 0 or 1.2 mg/ml
pepsin (providing a 20:1 wt/wt pepsin:A? ratio, or
roughly a 2.5:1 mol ratio, when both are present). To
mimic the timing of the on-line experiments, solutions
containing pepsin were generally mixed in an Eppen-
dorf vial using a vortex mixer so as to minimize reaction
time; ?20 s was the minimum practical reaction time.
The digestions were quenched by adding sufficient 0.2
M ammonium hydroxide to bring the final solution to
pH around 8. Following quenching, 100-?L aliquots
were analyzed by HPLC and HPLC/MS (details be-
low). The retention time of A? was around 15.0 min.
Hydrolysis products eluted in the range of 8 to 16 min.
High Performance Liquid Chromatography
Reversed-phase HPLC and LC-MS experiments were
conducted with an Agilent 1100 Series LC/MSD system
(Wilmington, DE) with a ZORBAX SB-C3 column (pore
size: 5 ?m, diameter: 3 mm, length: 150 mm), an
autosampler, serial UV and electrospray MS detectors,
and a splitter (splitting ratio: 100:1). The solvent flow
rate was 1 mL/min, and the temperature was controlled
at 25 °C. The solvents used were 0.1% formic acid in
water (Solvent A) and 0.1% formic acid in MeCN
(Solvent B). The column equilibrated to 1% Solvent B
before injection, and then the sample was eluted with a
linear gradient of 1 to 51% Solvent B over a 40 min
period. The separation was monitored by absorbance at
215 nm and by MS scans from m/z 300 to 2000.
The LC-MS experiments employed the electrospray
source operating in the positive ion mode. The drying
gas flow (N2), nebulizer pressure (N2), drying temper-
ature, and capillary voltage were set at 3 L/min, 35
psig, 300 °C, and 3000 V, respectively.
Electrospray Ionization MS
A Q-Star XL quadrupole time-of-flight hybrid instrument
(Applied Biosystems, Foster City, CA) equipped with an
IonSpray source was used to acquire electrospray ioniza-
tion (ESI) MS and MS/MS spectra not involving LC. All
experiments were performed in the positive ion mode.
Source parameters including the sprayer position were
optimized for high sensitivity and stability. The IonSpray
voltage and declustering potential were 4500 and 60 V,
respectively. Except as noted, the sprayer tip was posi-
tioned about 12 mm away from the curtain plate with a
lateral displacement about 5 mm off-axis at an angle of 45°
to the ion sampling orifice. A microammeter (Simpson
Electric Co., Chicago, IL) was connected between the high
voltage power supply and the ion source connector to
J Am Soc Mass Spectrom 2007, 18, 208–217TRIAXIAL ELECTROSPRAY PROBE COUPLED WITH HDX-MS
Ravindra Kodali for help with Figure 3. The authors are grateful to
Dr. Albert A. Tuinman for helpful discussions.
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