Y.O. Tsybin et al., Eur. J. Mass Spectrom. 8, 389–395 (2002) 389
CE and ECD FT-ICR MS for Peptide Mixture and Protein Digest Analysis
Y.O. Tsybin et al., Eur. J. Mass Spectrom. 8, 389–395 (2002)
Youri O. Tsybin and Per Håkansson
Division of Ion Physics, The Ångström Laboratory, Uppsala University, Box 534, SE 751 21, Uppsala, Sweden
Magnus Wetterhall, Karin E. Markides and Jonas Bergquist *
Department of Analytical Chemistry, Uppsala University, Box 531, SE 751 21, Uppsala, Sweden. E-mail: Jonas.Bergquist@kemi.uu.se
Recent advances in peptide fragmentation techniques and mass spectrometry have opened up the possibility of combining peptide separation techniques, such as capillary electrophoresis (CE) and capillary liquid chromatography, with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and electron capture dissociation (ECD) in order to characterize peptide mixtures and protein digests. The results presented in this study show that CE/ECD-FT-ICR MS can be employed for peptide characterization in mixtures of standard peptides and in peptides resulting from the enzymatic digestion of proteins. Furthermore, the technique has potential for the identification and localization of post-translational modifications in peptides and proteins.
Keywords: protein, peptide mixture, enzymatic digest, trypsin, capillary electrophoresis, mass spectrometry, Fourier transform ion cyclotron resonance, electron capture dissociation, sequencing, post-translational modifications
Introduction
Capillary electrophoresis (CE) is an important complementary technique to liquid chromatography (LC) due to its high separation efficiency, low sample consumption and ability to separate molecules based on their charge/size ratio in solution.
1
During recent years, mass spectrometry has grown to be one of the most commonly used detection techniques for compounds separated by CE. The coupling of CE to mass spectrometry was first reported using an electrospray ionization (ESI) interface.
2
Later on, the CE technique was coupled to continuous-flow fast-atom bombardment
(CF-FAB)
3 and matrix-assisted laser desorption/ ionization
(MALDI).
4
The coupling of CE to MS was recently reviewed by Wetterhall et al.
5
Despite the numerous advantages of the combination of CE and mass spectrometry (CE/MS) the technique has some drawbacks and fundamental limitations.
In the case of the ESI interface, difficulties can arise from high concentrations of buffer ions, a condition generally required for a good CE performance. Furthermore, the sample size is usually limited to about 2% of the capillary volume in order to circumvent the loss of separation efficiency.
This limitation can, in some cases, be overcome by on-column pre-concentration techniques.
6
Various mass analyzers have been employed in CE/MS analysis. The rapid full-mass-range acquisition speed of a time-of-flight (ToF) mass analyzer makes it ideally suitable for high efficiency CE separations.
7
The quadrupole mass analyzer has a slower acquisition rate than the ToF analyzer but offers tandem mass spectrometry (MS/MS) possibilities.
Mostly due to the latter advantage, the quadrupole is the most commonly used analyzer in combination with CE. The
Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer
8 has, in recent years, gained popularity for use with liquid-based separation techniques.
9
The FT-ICR mass spectrometer has an unsurpassed mass resolving power (>
1,000,000 FWHM) and offers multiple tandem mass spectrometry (MS n
) opportunities. The principal drawbacks of the FT-ICR mass analyzer are a rather slow scanning speed and a high running cost. Alternatively, the advantages of each mass analyzer can be combined and coupled to CE as in the recently developed hybrid mass analyzers.
DOI: 10.1255/ejms.514
ISSN 1356-1049 © IM Publications 2002

390 CE and ECD FT-ICR MS for Peptide Mixture and Protein Digest Analysis
Electron capture dissociation (ECD) is a mild peptide and protein fragmentation technique.
10
The method is based on the interaction of low energy (< 0.5 eV) electrons with ions trapped under high vacuum conditions in a Penning trap. Ion-electron reactions result in specific peptide backbone cleavages, which provide complementary peptide structure information and extensive sequence coverage. Up to date, the ECD technique has only been implemented in
FT-ICR mass spectrometry. De novo peptide sequencing
11 and the characterization of labile post-translational modifications
12-15 are typical application areas for ECD. Due to recent technique development, the reaction rate of the
ECD process has been increased by at least one order of magnitude.
16
The increase in the reaction rate makes it possible to employ ECD in time-limited applications. Previously, our groups have reported the combination of liquid chromatography and ECD FT-ICR mass spectrometry for the analysis of peptide and protein mixtures.
17
The performance and importance of the developed technique were confirmed by the results obtained simultaneously and independently in similar experiments by another group.
18
In the frame of the present study, ECD FT-ICR mass spectrometry has, for the first time, been applied for the on-line structural analysis of peptides and protein digests separated by capillary electrophoresis.
Experimental and methods
Reagents and materials
Acetonitrile (LiChrosolv, Merck, Darmstadt, Germany), methanol (LiChrosolv), acetic acid (pro analysi,
Merck, Darmstadt, Germany) and ammonium acetate (pro
analysi) were used to prepare the separation buffer. Water was obtained from a Milli-Q purification system (Millipore,
Bedford, MA, USA). Polyimide sealing resin used for capillary outer surface coating was obtained from Alltech
(Deerfield, IL, USA). Graphite powder (1–2
µ m particle size) used as an outer conductive coating material, and the inner surface capillary coating reagent [3-
(methacryloylamino)-propyl]trimethylammonium chloride
(MAPTAC) were both obtained from Aldrich (Milwaukee,
WI, USA). The reagent 7-oct-1-enyltrimethoxysilane was purchased from Fluka (Buchs, Switzerland). N,N,N
′
,N
′
tetramethylethylenediamine (TEMED) was purchased from
Bio-Rad (Hercules, CA, USA). Bare fused- silica capillaries were obtained from Polymicro Technologies (Phoenix, AZ,
USA). ZipTip
C18
®
Corporation.
pipette tips were purchased from Millipore
Samples and sample preparation
A mixture of standard peptides included substance P
(MW 1347), bombesin (MW 1670), melittin (MW 2845), angiotensin I (MW 1296), insulin chain B (MW 3495), bradykinin (MW 1060), and renin substrate (MW 1759). The peptides were diluted in water to a final concentration of
1 µM (except bradykinin, substance P and bombesin which were diluted to final concentrations of 20 µM, 10 µM and
5 µM respectively). The human serum albumin (HSA) tryptic digest concentration was equivalent to 5 µM HSA.
All peptides and proteins were purchased from Sigma (St.
Louis, MO, USA). The tryptic digestion of HSA and sample pre-treatment of the digest prior to CE analysis was conducted according to Palmblad et al.
19
Safety considerations
All acids and bases should be handled with great care.
The CE column coating reagents [3-(methacryloylamino)propyl]trimethylammonium chloride, 7-oct-1-enyltri methoxysilane and N,N,N
′
,N
′
-tetramethylethylenediamine are irritants, which should be handled with care using protective gloves. Acetonitrile is combustible, volatile and toxic if exposed to lungs or skin. To avoid an electrical shock, the high voltage power supplies should be handled with extreme
Figure 1. A schematic drawing of the experimental set-up for capillary electrophoresis/Fourier transform ion cyclotron resonance electron capture dissociation mass spectrometry.

Y.O. Tsybin et al., Eur. J. Mass Spectrom. 8, 389–395 (2002) 391 care. The peptides and HSA samples are biologically active and should be handled using protective gloves.
Mass spectrometry
The experimental set-up is presented schematically in
Figure 1. All experiments were performed using a 9.4 T
Bruker Daltonics APEX II FT-ICR mass spectrometer
(Bruker Daltonics, Billerica, MA, USA). A general description of the instrument and its performance characteristics has been published elsewhere.
20
Positively-charged ions were produced by electrospray ionization and trapped by the sidekick trapping technique in the ICR trap. No ion isolation was performed prior to fragmentation reactions.
For the mixtures of standard peptides, a total of 256 mass spectra of 128 k data points were acquired. Each recorded mass spectrum was a sum of several single mass spectra. The time,
τ
, required for acquisition of a single mass spectrum can be estimated using the following approximation:
τ ≈ t hexapole
+ t ion generation
+ t
ECD
+ t analysis where t hexapole is the time of ion storage in an external hexapole ion trap (typical storage time is about 0.6–1.0 s), t ion generation is the time (less than 0.01 s) required for ion extraction from the hexapole trap and transportation into the ICR cell in a magnetic field, t
ECD is the time for ion-electron interactions in the
ICR trap and t analysis is the time (about 0.2 s, depending also on the number of data points acquired) required for ion excitation, detection and data transfer to the workstation. The time required for electron irradiation was not longer than 0.2 s due to the use of the high rate ECD. The hexapole ion storage time was minimized by discarding the quenching event of the external ion trap. Thus, ions were accumulated in the hexapole ion trap during the time-consuming electron capture process and mass analysis time. This optimization made it possible to acquire a single scan mass spectrum every
0.4–0.5 s. The sensitivity of the instrument required summation of at least four single-scan mass spectra in order to obtain a signal-to-background ratio sufficient for subsequent data analysis. Therefore, the overall rate of the data acquisition was one mass spectrum every 2–4 s, which often is enough to effectively follow the elution of peptides from the CE capillary.
Capillary electrophoresis system
The CE system consisted of an in-house assembled CE set-up
21 including a high voltage power supply (Bertan,
Hicksville, NY, USA) providing a negative potential. Fusedsilica capillaries (25 µm i.d. × 360 µm o.d.) of 60 cm length were used for CE separations. The outlet ends of the capillaries were mechanically tapered
22 and the exposed bare fusedsilica was “Black Dust”
7 coated. The inner surface of all capillaries was derivatized with MAPTAC
23 to produce a positively-charged surface. In order to achieve a good compromise between migration time and resolution, the buffer was prepared taking into account considerations reported by Samskog et al.
24
The amount of organic modifier
(acetonitrile) was set to 25% (v/v) and the buffer system was
12.8 mM acetic acid + 0.2 mM ammonium acetate, corresponding to a pH of about 3. The buffer was filtered and degassed by ultrasonication. The capillary was equilibrated with 4–5 capillary volumes of buffer prior to injection. The samples were injected hydrodynamically at 10 psi for 10 s
(corresponding injected volume ~ 10 nL). The CE separation voltage was set to –30 kV, the electrospray emitter end was grounded and the potential at the mass spectrometer entrance orifice was set to –3 kV, yielding a field of
~ 500 V cm
–1
(corresponding current of about 40 µA).
Electron capture dissociation
Low-energy electrons were injected on-axis into the FT-
ICR ion trap from the side opposite to the ion entrance. The electron injection system was based on an indirectly- heated cathode with a large electron-emitting area.
16
The electron beam was 6 mm in diameter to maximize the overlap of ion and electron clouds in the FT-ICR ion trap. All ions in the ion trap were subjected to ECD conditions. Two types of experimental schemes were employed. In the first experimental sequence, the injection of electrons was switched on and off in alternate spectra, thus acquiring both CE/ECD-
MS and CE/MS data during a single two-dimensional data acquisition experiment.
In the second experimental sequence, CE/MS data was first acquired without ECD, followed by a consecutive run in CE/ECD-MS mode. The cathode heating current was varied between 2.0–2.4 A, the electron-emitting surface potential was varied between 0.4 –
0.6 V and the accelerating grid potential between 5–10 V.
Both end-plate potentials in the ion trap were set at 1.5 V relative to the cylindrical cell wall. The electron pulse duration was varied between 100–200 ms.
Data analysis
The ECD fragment ions were defined as those present only in ECD spectra. Fragment ions could then be identified using the precursor ion masses and by matching their electrophoretic peak profiles as generated by the XMASS
TM software chromatography module (Bruker Daltonics, Billerica,
MA, USA). Theoretical c- and z-ions were generated by the
SequenceEditor
TM application (Bruker Daltonics). For the analysis of the protein digest, the same program was used to predict the masses of enzymatic fragments and the corresponding ECD fragments.
After peak assignment for each mass spectrum, the generated peak lists and theoretically-predicted fragment masses were subjected for analysis by the BioTools
TM version 2.1 (Bruker Daltonics) data analysis software. The ECD peak assignment module of the program was modified in order to take into account all possible ECD fragments, including secondary ECD fragments, such as [c + H]
+
,
[z + H]
+
, [c – H
•
]
+
, [z – H
•
]
+
. Tandem mass spectra corresponding to a particular precursor ion were also summed in

392 CE and ECD FT-ICR MS for Peptide Mixture and Protein Digest Analysis
Figure 2. Selected ion electropherograms of peptides from the peptide mixture. 1—insulin chain B, 2—melittin, 3—bombesin,
4—renin substrate, 5—angiotensin I, 6—bradykinin, 7—substance P. Electron irradiation was switched on and off in alternate mass spectra.
order to increase the signal-to-background ratio and possibly detect more ECD fragments.
Results and discussion
The performance of the CE/ESI-FT-ICR MS system was first optimized, regarding resolution and analysis time, using a mixture of standard peptides. The CE peak widths were sufficiently broad for data acquisition in the alternating mode, thus ECD and non-ECD spectra were acquired during a single two-dimensional experimental run, similar to the algorithm presented by Palmblad et al.
17
A typical set of selected ion electropherograms is presented in Figure 2. The migration order of the peptides corresponds to increasing mass-to-charge ratio. The precursor peak intensities were, as expected, significantly reduced during electron irradiation.
The melittin ions did not give a signal that was intense enough for subsequent tandem mass spectrometry.
The structural analysis of larger proteins by LC FT-ICR mass spectrometry is commonly performed by employing tryptic digestion during the sample preparation step.
19
The resulting mixture of tryptic fragments contains mostly peptides of molecular weight 1–3 kDa depending on the digestion conditions. Tryptic peptides have a basic amino acid residue at the C-terminus. The concentrations of tryptic peptides are generally much lower in comparison with available peptide concentrations for standard peptide mixtures.
Hence, the electrophoretic peak width of each individual peptide is narrower and insufficient for the alternating ECD mode. Therefore, the experiments were performed twice on the same sample. First, a complete data set of 128 mass spectra was acquired without electron irradiation. During the consecutive run, low energy electrons were injected into the
ICR ion trap for all recorded spectra. As a result, two data sets were acquired that were matched regarding precursor and fragment ions.
Peptide mixture analysis
Peptide fragments of c- and z-types were identified as peaks only occurring during the ECD mode. A summary of the ECD-generated sequence tags for the peptides from the peptide mixture is presented in Figure 3. For peptides like bradykinin, the high content of proline (Pro) residues prevents an extensive backbone fragmentation. However, as will be mentioned below, by taking into account the presence of Pro residues, the sequence could be correctly identified using the tandem mass spectrometry data.
As can be seen in Figure 3, comprehensive sequence tags were generated only for peptides with a molecular weight below 2 kDa. Fragment peaks with masses above
1 kDa are not easily achievable. Therefore, the N-terminal side of peptides is, in most cases, represented by c-type fragment ions and the C-terminal side by z-type ions. The efficiency of ECD fragmentation and the fragment relative abundances strongly depend on the primary structure of the molecule.
25
The presence of Pro residues complicates the analysis of ECD fragmentation spectra. On the other hand, basic residues (His, Lys and Arg) facilitate the formation of
ECD fragments. Depending on the position of basic amino acids along the peptide backbone, either c- or z-type frag-
Figure 3. Observed backbone cleavages of peptides from the peptide mixture induced by electron irradiation. Each mass spectrum was recorded for 4 s.

Y.O. Tsybin et al., Eur. J. Mass Spectrom. 8, 389–395 (2002) 393
Figure 4. ECD mass spectrum of substance P acquired for 4 s.
De novo sequencing was made using BioTools software. Notation:
[M]—substance P, [N]—bradykinin.
ments are more stable and thus produce a detectable signal.
The results show that, for peptides with a sequence favorable for ECD, such as substance P, an efficient fragmentation spectrum can be obtained in a short time (less than 4 s). The information obtained is sufficient for de novo peptide sequencing. This fact is illustrated by Figure 4 with an ECD mass spectrum of substance P acquired during 4 s. This mass spectrum was submitted to the de novo sequencing procedure supported by the BioTools TM software. The search principle is based on fitting the distances between the peaks found in the mass spectra to known amino acid masses.
Using electron-induced fragmentation, a peptide backbone can not be cleaved at the N-terminal side of a proline residue.
Therefore, the mass of the resulting dipeptide (a pair of amino acids, of which one is proline), can be used for matching the distance between the found peaks. As can be seen in
Figure 4, the identified amino acid sequence corresponds completely to a large part of the primary structure of substance P, with the exception that isoleucine and leucine residues can not be distinguished. This problem can be solved by employing a new technique of “hot ECD”, 26 where electrons that are more energetic than in conventional ECD are used.
Tryptic digest analysis
Taking into account the results of the CE/ECD FT-ICR
MS analysis of the peptide mixture, described above, the application of ECD for the analysis of tryptic peptides separated by CE should focus on searching for the abundant mul-
Figure 5. A two-dimensional contour plot of the data set containing ECD mass spectra of 50 fmol HSA tryptic fragments separated by capillary electrophoresis. The electron irradiation was switched on during acquisition of all spectra. The isotopic clusters of (1) doubly-charged HSA [509–524] and (2) singly-charged HSA [427–434] fragments are shown in the inset.

394 CE and ECD FT-ICR MS for Peptide Mixture and Protein Digest Analysis
Figure 6. ECD spectrum of HSA [509–524] tryptic fragment (RPCFSALEVDETYVPK) obtained during 3 s with an electron irradiation time of 200 ms.
tiply-charged tryptic fragments with a sequence favorable for fragmentation by electron irradiation.
A part of a two-dimensional data set of tandem mass spectra is presented in Figure 5. To illustrate the performance of the CE separation a magnification of the region around m/z 950 is shown in the inset. The ordinate axis corresponds to the spectrum number. The isotopic clusters of doubly-charged HSA [509–524] and singly-charged HSA
[427–434] fragments are only present in consecutive mass spectra. This fact confirms that the narrow peak widths of the tryptic peptides rules out the possibility of alternately acquiring CE MS and CE MS/MS data in one run. The ECDgenerated sequence tag of the HSA [509–524] tryptic peptide is shown in Figure 6. The peptide mass, together with the sequence tag, were enough for protein identification in
Mascot database searches. In the inset the c
4
+ fragment ion is shown. The peak assigned by the number sign (#) is unknown.
The HSA tryptic fragment coverage obtained from a single CE FT-ICR MS run is shown in Figure 7. The incomplete sequence coverage can be explained by the fact that only peptides with an intensity above the intensity of a peptide shown in the inset were taken into consideration. However, regarding the fragment abundance and charge state, only a few of the observed tryptic fragments were found to be potential candidates for subsequent MS/MS analysis.
These are marked with an asterisk (*) at the side; ECD fragments were generated for all of them. Only sequence tags with an amino acid length of 3 and more are shown in Figure
7. The presented results were obtained with a 200 ms electron irradiation time and a spectrum acquisition time of
3–4 s.
Figure 7. Sequence coverage and a few of the ECD-generated sequence tags of HSA tryptic digest obtained by CE/ECD-FT-ICR
MS. The inset shows the doubly-charged ions of HSA
[469–490].
Conclusions
The technique of CE/ECD-FT-ICR MS has a potential for the analysis of peptide mixtures, including enzymatic digests of proteins. The benefits of a separation, such as CE, prior to mass spectrometric analysis are reduced ionsuppression and space-charge effects. Subsequent tandem mass spectrometry of the separated peptides is also facilitated due to a reduced number of precursor ions in each mass spectrum. The information gained from ECD could provide complementary structural information necessary for unambiguous protein identification. Complementary electropho-

Y.O. Tsybin et al., Eur. J. Mass Spectrom. 8, 389–395 (2002) 395 retic information can aid in the identification of peptides in mixtures or verify protein identification using peptide mass fingerprinting. The short experimental time required for
CE/ECD-FT-ICR MS (10–15 min) enables system optimization prior to analysis by LC/ECD-FT-ICR MS.
The results from CE/ECD-FT-ICR MS analysis of the
HSA tryptic digest show the wide application capabilities of the method. However, the sensitivity and still rather slow acquisition rate of the FT-ICR MS are, at this point, limiting the applicability of the method, for example, in the analysis of post-translational modifications. The forthcoming generation of FT-ICR mass spectrometers should feature a faster data acquisition rate, higher sensitivity and external in-flight ion isolation to improve the efficiency of tandem mass spectrometry of on-line separated peptide and protein mixtures.
Acknowledgments
Financial support from the Swedish Research Council
(Grant 13123), the Swedish Society for Medical Research and the Knut and Alice Wallenberg Foundation is gratefully acknowledged. JB has a senior research position from the
Swedish Research Council.
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Received: 16 October 2002
Accepted: 21 October 2002
Web Publication: 21 October 2002