Download Electronic excitation gives informative fragmentation of polypeptide

K.F. Haselmann et al., Eur. J. Mass Spectrom. 8, 117–121 (2002)
117
Fragmentation of Polypeptide Cations and Anions
K.F. Haselmann et al., Eur. J. Mass Spectrom. 8, 117–121 (2002)
Electronic excitation gives informative fragmentation
of polypeptide cations and anions
K.F. Haselmann, B.A. Budnik, F. Kjeldsen, M.L. Nielsen, J.V. Olsen and R.A. Zubarev*
Department of Chemistry, University of Southern Denmark, Campusvej 55, DK 5230 Odense M, Denmark
E-mail [email protected]
A Fourier transform mass spectrometer is a versatile instrument with a range of available fragmentation techniques. Comparison of
polypeptide fragmentation patterns revealed that the techniques involving electronic excitation, such as hot-electron-capture dissociation (HECD) and electron-detachment dissociation (EDD), are even more informative than vibrational excitation (VE) techniques
such as collisional activation. For dications of the peptide KIMHASELMANN, 11 eV HECD cleaved all inter-residue links in at least
two places, with up to five fragments characterizing each link. For dianions of the same molecule, VE produced only one backbone
cleavage whereas EDD gave ten, including five internal cleavage fragments. This is consistent with the general postulate that homogeneous electronic excitation yields more types of cleavage than near-equilibrium processes such as VE.
Keywords: polypetides, fragmentation, electronic excitation, ion–electron reactions, electron capture dissociation, collision-induced dissociation, electron-detachment dissociation
Introduction
V.L. Talroze belongs to the legendary generation of scientists who contributed to many important discoveries. His
paper with A.K. Ljubimova, published in 1952 with the
unpretentious title “Secondary processes in the Ion Source
of a Mass Spectrometer”, opened the era of organic ion–
molecule reactions,1 the probable mechanism by which the
universe synthesizes most of its organic matter. The battles
that V.L. Talroze had to withstand to defend this discovery
are also legendary (the methonium ion CH5+ still poses some
kind of a mystery for vibrational spectroscopy, with an infrared spectrum obtained only recently and not yet fully
2
explained). One of the lessons learned from that study was
that a mass spectrometer is a versatile source of information
on processes involving organic molecules.
Today, the attention of the scientific community has
shifted to larger biological polymers. Both natural and synthetic polypeptides and DNAs are increasingly not only
being used in traditional life science disciplines, such as biochemistry, molecular biology, neurobiology, pharmacology
and medicinal chemistry, but are also considered as potentially important building modules for nanotechnology.3 The
world of naturally-occurring polypeptide sequences is much
restricted compared to the vast realm of possible sequences.
However, polypeptide sequences do not have to be natural to
be biologically relevant. In our view, the term “biological” is
equally applicable to any polymer containing building
blocks found in living nature, just as the word “organic” is
applicable to any carbon-containing molecule. Therefore,
we consider any polypeptide to be biological, regardless of
origin or presence among known sequences in a protein or
DNA database.
For polypeptides of arbitrary sequence, tandem mass
spectrometry is of great importance for their characterization, since other techniques (for example Edman degradation) are not always efficient and/or exhaustive. Dissociation
in the gas-phase requires application of one of the available
activation methods. The range of the traditional techniques
that involve vibrational excitation (VE) is broad, from
collisional-activation dissociation (CAD) to infrared
dissociation (IRMPD or BIRD) to surface-induced
dissociation (SID). If we only consider multiply-charged
ions produced by electrospray ionization (ESI) and analyzed
by Fourier transform mass spectrometry (FT-MS), CAD
techniques would include nozzle-skimmer dissociation,
multipole-storage-assisted dissociation (MSAD),4 sustained
5
off-resonance irradiation (SORI) and electron-impact exci6
tation of ions from organics (EIEIO). In 1998, a new type of
reaction, electron-capture dissociation (ECD), was intro7
duced. More recently, two more ion-electron reactions have
been added: hot-electron-capture dissociation (HECD)8 for
9
polycations and electron-detachment dissociation (EDD)
for polyanions. All these techniques can be implemented in a
© IM Publications 2002, ISSN 1356-1049
118
Fragmentation of Polypeptide Cations and Anions
single FT instrument. Which technique is the most informative in sequence characterization of polypeptides? Here we
address this question by applying MSAD, SORI CAD, ECD,
HECD and EDD to the same molecule. More than a dozen
peptides were involved in the study: however, we illustrate
the general findings using one model peptide with the
sequence KIMHASELMANN. The length of the peptide (12
residues) and its amino acid composition is characteristic of
the synthetic peptide studies that our laboratory is involved
in.10
Experimental
Synthesis
The peptide was obtained using automated solid-phase
synthesis and the Fmoc (9-fluoronylmethoxycarbonyl) protection strategy on a research-scale ResPep peptide synthesizer (Intavis AG, Gladbach, Germany). After completion of
the synthesis, the resin was washed three times with
dichloromethane (Merck, Darmstadt, Germany) and dried in
a dry nitrogen flow for 15 min. Simultaneous cleavage of the
peptide from the resin and side-chain deprotection was performed at 21°C in a mixture (95 : 2.5 : 2.5, v/v) of
trifluoroacetic acid (Fluka, Steinheim, Germany),
triisopropylsilane (used as a scavenger) and MilliQ-water
(Millipore, CA, USA). After two hours of deprotection, the
peptide was precipitated with cold (0°C) t-butyl methyl ether
(Fluka). The peptide precipitate was redissolved in 1% aqueous acetic acid and lyophilized.
Mass spectrometry
Nano-electrospray mass spectrometry, using a
hexapole-based interface (Analytica of Branford, MA,
USA), modified with a heated metal capillary, was performed on a 4.7 T Ultima FT mass spectrometer from
IonSpec (Irvine, CA, USA). The peptide was dissolved in a
standard electrospray mixture of water, methanol and acetic
acid (49 : 49 : 2, v/v) all of the highest purity available. A
3–5 L aliquot was loaded into a metallized pulled-glass
capillary (MDS Proteomics, Odense, Denmark). Ions produced by electrospray were externally accumulated in the
hexapole for 0.5 s and transmitted to the FT cell by RF-only
quadrupole ion guides. Capture of ions in the open-ended
cylindrical cell with extra trapping plates was achieved by
gated trapping.
For performing dissociation experiments, ions of interest were isolated by application to the excitation electrodes
of a pre-programmed waveform. For SORI CAD, a 500 ms
RF-burst with an amplitude of 6 V was applied at a frequency approximately 2 kHz higher than that for the m/z
value of the molecular ions, together with a pulse of collision
gas (4 ms, N2 at 20 Torr). For electron-ion fragmentation
reactions (ECD, HECD and EDD), an indirectly-heated dis11
penser cathode operated at 5 V and 0.34 A was employed.
In the ECD experiments, the electron-emitting surface was
Figure 1. SORI CAD of dications of KIMHASELMANN (10 scans).
biased to –1 V for 200 ms and in HECD to –11 V for 250 ms.
In EDD of dianions, irradiation at 21 eV for 1200 ms was
used. MSAD in the hexapole of the electrospray interface
was carried out by increasing the external accumulation time
to three seconds.
Results and discussion
SORI CAD of dications
The SORI CAD spectrum of the 2+ ion is shown in Figure 1. The precursor ion was totally fragmented and the base
peak corresponds to the loss of water from the dication. Peptide bond fragmentation (b, y fragments) gave full sequence
coverage, with eight complementary pairs. Only one
2+
dicationic backbone fragment was observed, b11 . Among
singly-charged fragments, the most abundant N-terminal
+
+
fragment was b9 , while its complementary ion y3 was the
most abundant C-terminal product.
MSAD of positive ions
Compared with SORI CAD, fragment peaks in the
MSAD spectrum of dications were more abundant, with the
average m/z shifted to higher values (Figure 2), so that the
Figure 2. Multipole-storage-assisted dissociation of dications
of KIMHASELMANN (10 scans).
K.F. Haselmann et al., Eur. J. Mass Spectrom. 8, 117–121 (2002)
Figure 3. Electron-capture dissociation of dications of
KIMHASELMANN. Asterisks denote the second and third harmonics from the ICR-signal (100 scans).
cleavages of three out of four N-terminal backbone bonds
were not observed. No complementary pairs were obtained,
either. The relative peak intensities in the m/z range from 800
+
to 1200, however, remained the same, with the b9 product
being again the most abundant. The absence of low-mass y
ions in the MSAD spectrum could be due to the massdiscrimination effect of the gated trapping, but it could also
be an inherent feature of MSAD. For other molecules, the
tendency of MSAD to promote formation of ions with high
m/z was also noticed. MSAD does not employ precursor-ion
selection and it does not inhibit secondary fragmentations,
therefore, the resultant ion population is often dominated by
the most stable fragments (usually y ions). The ion stability
depends upon the intramolecular charge density,12,13 which is
higher for species with smaller m/z. Proton transfer to neutral
14
species occurs if accumulation time is long (which can
account for the presence of singly-charged ions in the
MSAD spectrum), with higher proton-transfer rates for
smaller ionic species.
ECD of dications
At low electron energies (<1 eV), direct electron capture by dications was promoted, with the resulting mass
spectrum given in Figure 3. Here, ECD cleaved all interresidue bonds. As is typical for ECD spectra, mostly N–Cα
•
bonds were broken to yield c and z ions. Since the intermediate species were singly-charged radical cations, no complementary fragments from N–Cα cleavage were observed. On
•
the other hand, the fact that no c-ions smaller than c4 or z
ions smaller than z9 were observed identified His4 as one of
the protonation sites. This was expected due to the high gasphase basicity of that residue. The position of the other
protonation site is less clear [ECD of n-cations usually
reveals the location of (n–1) charges]. Among c fragments,
the most abundant was c11; the peaks, due to the loss of the Cterminal amino acid, are usually intense in ECD spectra. No
•
losses of neutral groups were observed from c or z ions.
Additionally, two y species were observed at lower
intensities, which together with c ions gave two complemen-
119
Figure 4. Hot-electron-capture dissociation of dications of
KIMHASELMANN (200 scans).
+
tary pairs. Whereas y9 could be due to the minor fragmentation channel in ECD that gives y+ + a• + CO,15 the presence
2+
of the doubly-charged y10 ion can only be explained by the
excitation in inelastic collisions during electron irradiation
(EIEIO).
Here, ECD was not more informative than CAD. This is
a typical situation for <1.5 kDa species, with ECD becoming
relatively more important for larger species.
HECD of dications
When the electron energy was increased to 11 eV, the
spectrum character changed dramatically (Figure 4). Many
more fragments were observed, especially in the low m/z
region. Full sequence coverage was obtained with full
(within the detected m/z region >200 Th) b, c and y series;
every inter-residue link produced between two and five different fragments. All five z ions and six a ions were evenelectron species 1 Da below the expected mass for
homolytic cleavage, suggesting either heterolytic cleavage
or hydrogen atom loss upon homolytic backbone fragmentation. Secondary fragmentation is a common phenomenon in
hot-electron-capture dissociation (details of this regime are
8
given elsewhere).
The EIEIO effect was rather significant: the most abundant fragment ions were b ions in the higher m/z region,
whereas the most abundant fragments in the lower m/z
Figure 5. SORI CAD of dianions of KIMHASELMANN (10 scans).
120
region were y ions. Of the eleven b ions, ten lost water. But,
even if only single-bond cleavages were taken into account,
HECD was easily the most informative fragmentation technique for positive ions.
SORI CAD of dianions
In the negative-ion mode, abundant molecular dianions
of the peptide were observed. However, SORI CAD produced mostly losses of small neutral groups (Figure 5) with
only one backbone fragment, b11. MSAD did not give additional cleavages. This result illustrates the difficulty of
obtaining structural information from peptide anions by VE
techniques. Only a few studies that report on tandem mass
spectrometry (MS/MS) of deprotonated peptides larger than
16
five residues have been published so far, despite the fact
that most modern mass spectrometers with MS/MS capabilities can easily handle ions of both polarities. At the same
time, many peptides are acidic and ionize far better in the
negative-ion mode. In addition, some labile modifications,
notably sulfation and phosphorylation, promote anion formation.
EDD of dianions
21 eV electron irradiation of dianions produced an oxi–•
dized radical ion [M–2H] which fragmented to give nearly
complete sequence coverage except for one backbone bond
between Ala5 and Ser6 (Figure 6). Despite the fact that Metoxidized species were not perfectly removed from the ICR
cell during isolation of the precursor dianions, their fragments were of low abundance and did not interfere with the
rest of the spectrum. The most abundant fragment peak was
due to carbon dioxide loss from the oxidized radical species.
This is readily explained by the ionization of negativelycharged carboxylic groups (for example, Glu7 or the C-ter•
minus). This gives an RCO(O ) group, the radical site of
17
which initiates α-cleavage with carbon dioxide loss. Upon
backbone ionization, backbone fragmentation occurred due
9
to electron-hole recombination. Interestingly, five internal
Fragmentation of Polypeptide Cations and Anions
fragments were observed, a sign of high energy deposition
by the electrons.
Vibrational vs electronic excitation
The much greater variety of fragmentation channels in
HECD of dications and EDD of dianions, compared with
CAD of the same species, requires explanation. Of course,
the fragmenting species in both techniques are different:
they are radical ions in the first case and even-electron species in the latter case. However, stable polypeptide radical
cations tend to produce less backbone fragmentation than do
the corresponding even-electron ions.18,19 Therefore, another
explanation is needed, and we offer one in the form of a simple model. According to this model, near-equilibrium excitation in CAD leaves the ions primarily in the ground
electronic state, with N0 parallel channels open for dissociation at a given level of the excess energy. Electronic excitation by electron impact is rather homogeneous (no strongly
“chromophoric” sites as in UV photoexcitation). Therefore,
the ion can be excited to multiple (n) electronic states, where
n is at least of the order of L/λ, where L is the peptide length
in Å and λ is the electron localization length in polypeptides.
The value of λ can be estimated as ≤6–10 Å.20,21 The lifetime
of first excited electronic states in polypeptides is of the
order of nanoseconds, with much longer lifetimes for high
Rydberg states.22 This is enough for single-bond cleavage.
Each of these states has Ni parallel channels for dissociation
(i = 1,..n). In general, Ni ≈ N0. Although some or even many
of these channels are the same as in the ground electronic
state, the sum ΣNi, i = 0..n, is always larger than N0: it cannot
be smaller since N0 is also included in it. Therefore, homogeneous electronic excitation should lead to a more diverse
(informative) fragmentation than vibrational excitation at
the same energy excess. Among the new channels opened by
electronic excitation, the model predicts the dominance of
single-bond cleavages, since losses of neutral molecules are
too slow (require rearrangement) to compete with
intramolecular energy conversion that removes electronic
excitation. This, however, does not preclude slow secondary
fragmentation occurring after the product separation.
Conclusions
Figure 6. Electron-detachment dissociation of dianions of
KIMHASELMANN (200 scans).
We have demonstrated with one typical example that
the most informative fragmentation techniques in FTMS are
those involving electron-ion reactions with electron energies
sufficient to produce electronic excitation. Experiments with
other tested molecules were in agreement with this observation. A simple model is proposed that explains the effect
through the existence of multiple electronic excitation states,
each of them with a set of parallel fragmentation channels.
K.F. Haselmann et al., Eur. J. Mass Spectrom. 8, 117–121 (2002)
121
Acknowledgement
Heinrich Gausepohl is gratefully acknowledged for help
in setting up the peptide synthesis facility. The work was
funded by the Danish research councils (SNF, grant number
51-00-0358; STVF, grant number 0001242). The FTMS
instrument was funded by the Instrument Center Program
(grant number 9700471).
12.
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Received: 9 November 2001
Revised: 7 January 2002
Accepted: 19 February 2002
Web Publication: 8 April 2002