In-spray supercharging of peptides and proteins in electrospray ionization mass spectrometry.

Abstract

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.

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In-Spray Supercharging of Peptides and Proteins in Electrospray
Ionization Mass Spectrometry
Saša M. Miladinović,†Luca Fornelli,†Yu Lu,‡Krzysztof M. Piech,†Hubert H. Girault,‡
and Yury O. Tsybin*,†
†Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland
‡Laboratoire d’Electrochimie Physique et Analytique, Station 6, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne,
Switzerland
*
S Supporting Information
ABSTRACT: Enhanced charging, or supercharging, of
analytes in electrospray ionization mass spectrometry (ESI
MS) facilitates high resolution MS by reducing an ion mass-to-
charge (m/z) ratio, increasing tandem mass spectrometry
(MS/MS) efficiency. ESI MS supercharging is usually achieved
by adding a supercharging reagent to the electrospray solution.
Addition of these supercharging reagents to the mobile phase
in liquid chromatography (LC)-MS/MS increases the average
charge of enzymatically derived peptides and improves peptide
and protein identification in large-scale bottom-up proteomics
applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and
proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a
dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into
the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation
from a droplet in the ESI ion source.
E
due to its ability to generate multiply charged ions.1Specifically,
multiple charging facilitates the observation of large molecular
ions with narrow m/z range mass analyzers. The commonly
employed low-to-medium resolution ion trap mass spectrom-
eters (IT MS) are typically limited by 4000 m/z upper m/z
threshold. High resolution mass spectrometers, for example,
Fourier transform mass spectrometers (FTMS), can operate at
substantially higher m/z range. However, FTMS resolution
rapidly reduces with increasing m/z and, additionally, modern
FTMS platforms are often hybridized with IT MS, including
quadrupole MS, that further limit their working m/z range.2
Enhanced charging, or supercharging, of biomolecules
increases the charge (z) of the ions observed in ESI MS.3−9
Furthermore, higher charge states of selected precursor ions
increase efficiency of tandem mass spectrometry (MS/MS),
especially electron-based MS/MS, including electron capture
dissociation (ECD) and electron transfer dissociation
(ETD).10−13Typically, the supercharging in ESI MS is
achieved by adding a small amount of a supercharging reagent
to the analyte solution.3−7,9The most commonly employed
supercharging reagents are m-nitro benzyl alcohol (m-
NBA),4−7,9,14−17dimethyl sulfoxide (DMSO),5,18,19and tetra-
methylene sulfone (sulfolane).7,20−22The addition of small
amounts of supercharging reagents to the mobile phase in
liquid chromatography tandem mass spectrometry (LC-MS/
lectrospray ionization mass spectrometry (ESI MS) allows
structural analysis of large intact biomolecules, primarily
MS) has been shown to increase the average charge state
distribution of enzymatically derived peptides, leading to
improved peptide and protein identification in large-scale
applications.23However, the presence of supercharging
reagents in the LC mobile phase influences the retention
time of the analytes and decreases chromatographic reso-
lution.18,23
The complete mechanism of the supercharging phenomenon
remains uncertain. Initially, it was proposed that an increase in
ESI droplet surface tension, as the result of the supercharging
reagent, played the main role in the supercharging process.5
Due to their low volatility, supercharging reagents enrich the
mature ESI droplet, thereby increasing surface tension.
According to Rayleigh charge limit theory, the surface tension
is directly proportional to charge availability of the droplet.5
However, other works on supercharging mechanism proposed
that, in the absence of conformational changes, supercharging is
independent of surface tension.24,25A study on supercharging
of proteins from their native solution by the Loo group claimed
that supercharging does not depend on the conformational
changes of proteins in the ESI droplet.25On the contrary, the
Williams group suggested that the conformational changes of
proteins are important in the supercharging process.6,15−17
Received:
Accepted:
Published: May 4, 2012
March 27, 2012
May 4, 2012
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Here, we demonstrate the possibility of increasing the
average charge state distribution of peptides and proteins by
introducing supercharging reagents directly into the electro-
spray’s Taylor cone (referred to as in-spray supercharging). The
presented methodology has a strong potential for application in
bottom-up, middle-down, and top-down LC-MS/MS experi-
ments, where it can be employed to increase the charge states
of the observed peptides and proteins without influencing
chromatographic separation.
■METHODS
Cytochrome C, myoglobin, and substance P were obtained
from Sigma-Aldrich (Buchs, Switzerland). For direct sample
injection, analytes were dissolved in water/methanol (50/50,
V/V) with addition of 0.1% formic acid to make 5 μM
solutions. The composition of the solvent mimics the
composition of the mobile phase that could be used to elute
the analytes in LC-MS experiments. The supercharging
reagents, including m-NBA, DMSO, and sulfolane were
purchased from Sigma-Aldrich (Busch, Switzerland) and
dissolved in water/methanol (50/50, V/V) to make the
commonly employed in supercharging ESI MS 0.5% m-NBA,
5% DMSO, and 1% and 5% sulfolane solutions.
To accomplish peptide and protein in-spray supercharging, a
dual-sprayer ESI microchip was employed.26−28The microchip
consists of two independent microchannels located on the
opposite sides of a chip. The microchannels are crossed at the
emitter tip on the top of each other but do not have a direct
connection. Therefore, the two solutions infused through these
channels can only mix in the Taylor cone upon the applied
voltage.26The dual-sprayer ESI microchip was coupled to a
hybrid linear ion trap Fourier transform ion cyclotron
resonance MS (LTQ FT-ICR MS, Thermo Scientific, Bremen,
Germany) equipped with a 10 T superconducting magnet
(Oxford Nanosciences, Oxon, UK).29The MS experiments
were performed with initial ion detection in the medium
resolution LTQ MS and validated with high resolution FT-ICR
MS ion detection. Electron capture dissociation (ECD) and
infrared multiphoton dissociation (IRMPD) were performed in
the FT-ICR MS,29whereas collision induced dissociation
(CID) was performed in the LTQ MS.
Analyte solutions were electrosprayed via one microchannel
of the dual-sprayer ESI microchip at a 200−500 nL/min flow
rate, whereas either water/methanol solution (50/50, V/V; for
nonsupercharging experiments) or the supercharging reagent-
containing solution was cosprayed through the second
microchannel with identical flow rate unless otherwise stated.
The ESI potential was applied directly only to the analyte
solution through the dedicated electrode, whereas the super-
charging reagent solution would experience the applied
potential only after mixing with the analyte solution in the
Taylor cone. We noted that the lifetime of a dual-sprayer ESI
microchip was longer when methanol was used as opposed to
acetonitrile. Therefore, the microchip materials should be
optimized to provide a longer life operation when acetonitrile is
used as a solvent in LC-MS experiment. The mass spectrometer
capillary temperature was kept at 200 °C, and the electrospray
voltage was set to 1.9 kV. The parameters used to describe
analyte charge states include the average charge state
distribution (zavg), the charge state of highest abundance peak
in the mass spectrum (zbase), and the maximum observed
charge state (zmax).5
■RESULTS AND DISCUSSION
Myoglobin and cytochrome C proteins contain 154 and 105
amino acids, respectively. Proteins of similar lengths are usually
targeted by top-down and middle-down proteomics.30
Previously, myoglobin and cytochrome C were successfully
in-solution supercharged with various reagents and meth-
ods.3−8,14,17−19,22The ESI mass spectrum of myoglobin ions
formed from a regular ESI solution (0.1% formic acid in
methanol/water (50/50, V/V) solution) showed a zavgof 18.8,
a zbaseof 19+, and a zmaxof 27+, Figure 1A. During this control
experiment, the supercharging solution sprayed from the
second channel was water/methanol (50/50, V/V). In-spray
supercharged myoglobin using 0.5% solution of m-NBA had a
Figure 1. LTQ FT-ICR MS of myoglobin when its solution was electrosprayed via one channel of the dual-sprayer ESI microchip whereas (A)
water/methanol, (B) 0.5% m-NBA, (C) 5% DMSO, and (D) 1% sulfolane were sprayed through the second microchannel.
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zavgof 21.7, a zbaseof 23+, and a zmaxof 31+, Figure 1B. The
effect of DMSO supercharging was comparable to the results of
m-NBA supercharging: the addition of DMSO increased zavgto
21.8, zbase to 20+, and zmax to 29+, Figure 1C. The
supercharging with sulfolane produced myoglobin mass spectra
abundant with sulfate adducts, Figure 1D. Protein sulfate
adducts in sulfolane in-solution supercharging were reported
previously.22
The influence of sulfolane quantity mixed with myoglobin
within the Taylor cone on the ion abundance is shown in
Figure S1, Supporting Information. The formation of sulfate
adducts influenced the signal abundance of myoglobin ions in
an inverse manner to the sulfolane concentration. At the same
time, the intensity of the heme signal was constant because
sulfate adducts were not observed with the heme ion. Besides
adduct formation, the presence of sulfolane increased zavgto
23.6, zmaxto 30+, and zbaseto 26+.
The efficiency of ion supercharging is protein dependent;
therefore, even proteins of a comparable size (e.g., myoglobin
and cytochrome C) may demonstrate very different super-
charging tendencies. Naturally, efficient supercharging of larger
proteins, e.g., transferrins and immunoglobulins, is the desired
outcome of ion supercharging applications. However, the
current development of even a solution-based ion super-
Figure 2. LTQ FT-ICR MS of cytochrome C when its solution was electrosprayed via one channel of the dual-sprayer ESI microchip whereas (A)
water/methanol, (B) 0.5% m-NBA, (C) 5% DMSO, and (D) 1% sulfolane were sprayed through the second microchannel.
Figure 3. LTQ FT-ICR MS of substance P when its solution was electrosprayed via one channel of the dual-sprayer ESI microchip whereas (A)
water/methanol, (B) 0.5% m-NBA, (C) 5% DMSO, and (D) 1% sulfolane were sprayed through the second microchannel. Asterisks denote
spontaneous chemical decomposition products, not originated due to the in-spray reaction with the supercharging reagents.
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charging method and technique, does not allow for efficient
supercharging of large intact proteins. Presumably, this decrease
in supercharging efficiency results from the significantly higher
extent of structure folding in larger proteins. Furthermore,
separation efficiency of proteins by liquid chromatography is
underdeveloped for large proteins, whereas for peptides and
small, 10−20 kDa, proteins, considered in the current work, LC
separation is well established. Therefore, the in-spray super-
charging developed here primarily targets bottom-up and
middle-down proteomics, where enzymatically produced
peptides of up to 20 kDa might have different ionization and
fragmentation capabilities compared to myoglobin and
cytochrome C. An extension of in-spray ion supercharging to
larger proteins would require further developmental work.
In-spray supercharging of cytochrome C demonstrated
similar behavior to both myoglobin in-spray supercharging
and cytochrome C in-solution supercharging, Figure 2.
Specifically, cytochrome C electrosprayed from a nonsuper-
charging solution showed a zavgof 14.6 with a zbaseof 14+ and a
zmaxof 20+, Figure 2A. In-spray supercharged cytochrome C
with m-NBA showed a zavgof 18.4 with a zbaseof 20+ and a zmax
of 23+; with DMSO a zavgof 17.5 with a zbaseof 20+ and a zmax
of 21+; and with sulfolane a zavgof 18.3, a zbaseof 19+, and a
zmax of 23+, see Figure 2B,C,D, respectively. In-spray
supercharged cytochrome C with DMSO showed mass
spectrum with a bimodal charge distribution, Figure 2C,
whereas sulfolane caused sulfate adduct formation, Figure 2D.
The origin of the bimodal charge distribution in Figure 2C is
presumably due to the variation of DMSO concentration
during ESI droplet evolution. It is known that supercharging
efficiency of DMSO depends on its concentration in the analyte
solution and ESI droplet.19
Substance P is a peptide consisting of 11 amino acids, a
length comparable with proteolytic peptides generated in
bottom-up proteomics experiments.27In a regular ESI MS
experiment, substance P showed a zavgof 2.4 with the zbaseof
2+, whereas in-spray supercharged substance P with m-NBA
had a zavgof 2.8 with a zbaseof 3+, and supercharged DMSO had
a zavgof 2.3 with a zbaseof 2+; see Figure 3A,B,C, respectively.
The zmaxwas 3+ in all experiments with substance P. When
DMSO was used as a supercharging reagent, a DMSO adduct
was observed exclusively on the triply charged ion, [Substance
P + DMSO + 3H]3+, Figure 3C. The adduct signal was
observed only when the capillary temperature of the MS was
colder than 200 °C and was easily lost upon very mild
vibrational activation, indicating the noncovalent nature of
adduct formation. Nevertheless, [Substance P + DMSO +
3H]3+species were sufficiently stable to be isolated in the LTQ
for CID-based MS/MS and transferred intact to the ICR ion
trap for IRMPD and ECD-based MS/MS. However, even
application of soft ion activation and dissociation conditions has
not resulted in product ions containing the adduct (data not
shown), further confirming the noncovalent nature of the
complex. When substance P was supercharged with sulfolane,
the zavgwas 2.8 and the zbasewas 3+, Figure 3D. However, the
signal intensities of 2+ and 3+ ions decreased significantly,
whereas sulfate adducts were not observed. Note, that the
observed spontaneous decomposition products shown in
Figure 3D are not the result of in-spray supercharging reaction
but arise from the sample decomposition in solution, which is
known to be substantial for substance P, from a separate
experiment without supercharging (data not shown).
The results reported in Figures 1−3 nicely demonstrate the
shift in the charge states observed upon in-spray addition of
supercharging reagents but do not directly address the impact
of supercharging on the overall signal magnitude. Indeed, the
benefit of in-spray or in-solution supercharging for ECD/ETD-
based middle-down and top-down proteomics could be
counterbalanced by a possible signal intensity drop due to a
supercharging reaction. First, appearance or substantial
intensity increase of ions with charge states higher than the
present without supercharging reaction are beneficial even if
intensity of lower charge state ions drops. Indeed, fragmenta-
tion efficiency of peptide and protein ions, especially by ETD
and ECD, drastically depends on the precursor ion charge state.
As such, even a moderate increase in charge state may
significantly improve the obtained sequence coverage. Second,
signal abundance variation can be compared for lower charge
state ions, including ions corresponding to the average charge
state and most abundant ions. Here, we observed that the signal
intensity of ions detected in FTMS is not dramatically affected
by the in-spray addition of supercharging reagents; the variation
is always within an order of magnitude. m-NBA and DMSO can
even cause a moderate signal increase, whereas the impact of
sulfolane on the signal magnitude was always negative under
the applied experimental conditions, including the employed
concentration levels of supercharging reagents (see Supporting
Information Tables S1 and S2 for more details).
■CONCLUSIONS
To improve the performance of LC-MS/MS-based peptide and
protein structure analysis, we developed an in-spray ESI
supercharging method. To accomplish the in-spray super-
charging, we employed a dual-sprayer ESI microchip containing
two microchannels ending near each other at the tip of the
emitter, which allows mixing of the two liquids within the
Taylor cone. Our results include successful demonstrations of
in-spray supercharging using the direct infusion of proteins,
cytochrome C and myoglobin, and a peptide, substance P, with
three commonly employed supercharging reagents, m-NBA,
DMSO, and sulfolane. The effect of the in-spray supercharging
method using dual-sprayer ESI microchip is comparable to
previously reported results where supercharging reagents were
added directly into electrospray solutions. It was found that m-
NBA-based in-spray supercharging abilities were superior to
both DMSO and sulfolane, producing not only slightly higher
average and most abundant charge states but also fewer
adducts.
Overall, the reported results demonstrate that peptide and
protein supercharging processes require a short interaction time
between analytes and supercharging reagents. On the basis of
the previously suggested dynamics of ESI droplet evolution in
time and considering that supercharging reaction takes place in
the droplet, the interaction time can be estimated as less than
1−2 ms when low flow rate ESI conditions are realized, as
performed in the current work.31,32Importantly, the analytes in
the online LC-MS/MS experiments, presumably, could be
supercharged once eluted from the chromatographic column
without influencing chromatographic performance. This opens
an attractive prospect for supercharging applications in routine
LC-MS/MS, including bottom-up, middle-down, and top-down
protein analysis.
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■ASSOCIATED CONTENT
*
Influence of sulfolane concentration on myoglobin peak
intensities. Myoglobin and substance P signal intensity variation
during in-spray supercharging with m-NBA, DMSO, and
sulfolane. This material is available free of charge via the
Internet at http://pubs.acs.org.
■AUTHOR INFORMATION
Corresponding Author
*Address: EPFL ISIC LSMB, BCH 4307, 1015 Lausanne,
Switzerland. E-mail: yury.tsybin@epfl.ch.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
We are grateful to Prof. Evan R. Williams for the discussions
and Matthew Wodrich for the comments on the manuscript.
This work was supported by the Swiss National Science
Foundation (project 200021-125147/1) and the Joint Research
Project of Scientific & Technological Cooperation Program
Switzerland-Russia (grant agreement 128357 between EPFL
and INEPCP RAS).
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