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TUTORIAL REVIEW
Cite this: Chem. Soc. Rev., 2013,
42, 5014
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Principles of electron capture and transfer dissociation
mass spectrometry applied to peptide and protein
structure analysis
Konstantin O. Zhurov, Luca Fornelli, Matthew D. Wodrich, Ünige A. Laskay and
Yury O. Tsybin*
This tutorial review describes the principles and practices of electron capture and transfer dissociation
(ECD/ETD or ExD) mass spectrometry (MS) employed for peptide and protein structure analysis. ExD MS
relies on interactions between gas phase peptide or protein ions carrying multiple positive charges with
either free low-energy (B1 eV) electrons (ECD), or with reagent radical anions possessing an electron
available for transfer (ETD). As a result of recent implementation on sensitive, high resolution, high mass
accuracy, and liquid chromatography timescale-compatible mass spectrometers, ExD, more specifically, ETD
Received 23rd November 2012
DOI: 10.1039/c3cs35477f
MS has received particular interest in life science research. In addition to describing the fundamental
aspects of ExD radical ion chemistry, this tutorial provides practical guidelines for peptide de novo
sequencing with ExD MS, as well as reviews some of the current capabilities and limitations of these
www.rsc.org/csr
techniques. The merits of ExD MS are discussed primarily within the context of life science research.
Key learning points
(1) Electron capture and transfer dissociation (ECD/ETD) are electron-mediated radical-driven techniques applied in tandem mass spectrometry (MS/MS) for
the structural analysis of peptides and proteins. Analytical and practical considerations of ECD/ETD MS/MS are presented and compared.
(2) ECD/ETD reactions lead to peptide backbone cleavage primarily at N–Ca bonds, whereas labile post-translational modifications, e.g., phosphorylation and
glycosylation, may remain intact. Proposed mechanisms of N–Ca bond cleavage are summarized.
(3) Peptide ECD/ETD mass spectra interpretation is explained step-by-step. Worked examples are provided to guide the reader through the complex sea of ECD/
ETD product ions, populated by radical and even-electron ions, as well as ions containing chemically-modified amino acid residues.
(4) Are ECD and ETD MS/MS similar in terms of fragmentation patterns provided? Direct comparison is provided for fragmentation of a peptide and a protein,
and notable differences between the two methods are shown and explained by considering the parameters of the corresponding experimental set-ups.
(5) ECD/ETD MS/MS applications in life science research are briefly discussed: how the unique characteristics of ECD/ETD make these MS/MS methods
favourable for analyzing modified peptides and proteins, as well as for structural analysis of protein–protein complexes beyond their primary structure,
specifically through isotopic labeling-based methodologies.
1. Introduction
Molecular mass is an intrinsic parameter that provides information on the identity of a molecule. Mass spectrometry (MS) is
an analytical technique that allows for accurate mass measurements even from very small amounts of complex samples
containing analytes covering a wide range of molecular sizes
and concentrations.1 In addition to the molecular weight
measurement of an intact species of interest, deduction of
certain structural characteristics is possible via the use of
Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de
Lausanne, EPFL ISIC LSMB, BCH 4307, 1015 Lausanne, Switzerland.
E-mail: yury.tsybin@epfl.ch
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Chem. Soc. Rev., 2013, 42, 5014--5030
tandem mass spectrometry (MS/MS).2 The latter enables isolation of charged species of interest within a specific mass/charge
(m/z) ratio window, their subsequent fragmentation and,
finally, acquisition of m/z values of the resulting fragment ions.
There are a number of MS/MS techniques implemented on
modern mass spectrometers.2–4 This review focuses on structural
analysis of peptides and proteins employing low-energy electronbased MS/MS fragmentation techniques – electron capture dissociation (ECD) and electron transfer dissociation (ETD).
Since the introduction of ECD MS in 1998,5 and of ETD
several years later,6 the specificity and mechanism of peptide
backbone bond cleavage in the gas-phase following ion–electron
interactions have been intensely studied.2,7 In contrast to the
classical slow-heating activation methods such as collision
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induced dissociation (CID), ExD cleaves peptide backbone N–Ca and
disulphide bonds, whilst preserving the labile post-translational
modifications (PTMs) to a substantially greater extent.2,8 Despite
its more recent development, ETD is more often employed than
ECD for peptide and protein structure analysis. The main reason is
the implementation of efficient ETD MS/MS on relatively affordable, robust and widespread ion trap mass spectrometers, which
deliver proteomics-grade performance, especially when coupled
with high resolution mass analyzers, such as Orbitrap FTMS or
time-of-flight (TOF) MS.9,10 In contrast, ECD has received broad
commercial implementation only on Fourier transform ion cyclotron
resonance mass spectrometers (FT-ICR MS), which are powerful
instruments, but are more complex to use and maintain.4
In this tutorial, we begin by considering applied, analytical,
and fundamental facets of ion–electron and ion–ion interactions for ECD and ETD (further referred to as ‘‘ExD’’) MS/MS
of peptides and proteins. We then discuss data analysis strategies specific for ExD MS/MS and provide two worked examples
illustrating peptide de novo sequencing using experimental ExD
mass spectra of an unmodified peptide and that of a phosphopeptide. We conclude by discussing selected applications of
ExD MS/MS in life science research.
2. Analytical aspects of ECD/ETD mass
spectrometry
2.1. Peptide and protein fragmentation concept and
nomenclature
Peptides and proteins are chains of amino acids (NH2–CaRH–
COOH, where R is one of 20 common amino acid side-chains)
joined by peptide bonds (C–N bonds) formed by a condensation
reaction between two amino acids. Fragmentation in MS/MS
targets one or more of the three constituent backbone bonds:
C–N (peptide bond), Ca–C, and N–Ca, Fig. 1. Interpretation of
the MS/MS spectra is primarily based on analyzing sequencespecific fragment ions formed by cleavage of these bonds. The
fragment ion type is identified by a letter, based on the type of
Konstantin O. Zhurov received
his MChem from the University
of Oxford, UK. He began his PhD
studies in fundamental and
applied mass spectrometry in
2011 under the supervision of
Prof. Yury O. Tsybin.
Konstantin O. Zhurov
Luca Fornelli
Dr Matthew D. Wodrich received
a BSc degree from the University
of Arizona (Tucson, AZ, USA) and
a PhD in computational/physical
organic chemistry from the
University of Georgia (Athens,
GA, USA) working with Prof.
Paul v. R. Schleyer. He is
currently
a
postdoctoral
researcher at EPFL where he
uses computational techniques
to investigate chemical problems.
Matthew D. Wodrich
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Luca Fornelli obtained his MSc in
Industrial Biotechnologies from
the University of Padova, Italy.
He is completing his PhD under
the supervision of Prof. Yury O.
Tsybin at EPFL with a project
focused on characterization of
large biomolecules by top-down
mass spectrometry.
The Royal Society of Chemistry 2013
Dr Ünige A. Laskay obtained her
MS
degree
in
Analytical
Chemistry from Babes- -Bolyai
University, Cluj, Romania, in
2008.
She
completed
her
doctoral
studies
at
Ohio
University,
USA,
in
the
laboratory of Prof. Glen P.
Jackson in the field of tandem
mass spectrometry method and
technique development. During
her first appointment as a
postdoctoral scholar in the
Ünige A. Laskay
laboratory of Prof. Vicki H.
Wysocki at the University of Arizona, she focused on the
application of mass spectrometry in life sciences research. Since
2011 she has been a postdoctoral scholar at EPFL, where her main
focus is on development of middle-down proteomics.
Chem. Soc. Rev., 2013, 42, 5014--5030
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denoted by ‘‘ ’’ (pronounced ‘‘dot’’), or an even-electron
species, denoted by ‘‘ 0 ’’ (pronounced ‘‘prime’’). Note that, for
the latter, the superscript is used only when there is a hydrogen
atom gain in comparison to homolytic bond cleavage.
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e capture
½M þ nHnþ þ e ð1 10 eVÞ ƒƒƒƒƒ!
(1)
bond cleavage
½M þ nHðn1Þþ ƒƒƒƒƒƒ! fragments
Fig. 1 Peptide backbone fragmentation: peptide structure, selected tandem mass
spectrometric (MS/MS) techniques, and fragment ion nomenclature. Electronmediated techniques for anionic analytes are highlighted in red.
backbone bond cleaved and which terminus the fragment
contains; the subscript indicates the number of amino acid
residues contained in the fragment, e.g., c4 or z10. An amino
acid residue entity comprises an amino acid without a water
molecule which is lost upon peptide bond formation.
Cleavage of the C–N (peptide) bond can be achieved by
vibrational activation of a peptide ion by collisions with neutral
gas molecules, known as CID, or by multiple photon-induced
activation and fragmentation, known as infrared multiphoton
dissociation (IRMPD), Fig. 1.3 On the other hand, low energy,
B1 eV, electron capture by multiply charged peptide cations in
ExD primarily results in cleavage of the N–Ca bond, forming
even-electron c 0 -ions (containing amino acid residues starting
from the N-terminus) and odd-electron radical z -ions (composed
of amino acid residues starting from the C-terminus), Fig. 1 and
eqn (1).2 The superscript indicates whether the ion is a radical,
Prof. Yury O. Tsybin received his
PhD degree in ion physics in 2004
(Uppsala University, Sweden)
working
with
Prof.
Per
Hakansson. The topic of his PhD
work was development of high
rate
electron
capture
dissociation mass spectrometry.
For the next 2 years he was a
postdoctoral research associate
with Prof. Alan G. Marshall at
the National High Magnetic
Field Laboratory in the USA
Yury O. Tsybin
focusing on applications of highresolution and tandem mass spectrometry. Since 2006 Tsybin has
been an assistant professor of physical and bioanalytical chemistry
at EPFL. In 2011 he received an ERC Starting Grant to develop the
super-resolution mass spectrometry.
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Due to charge neutralization following electron capture/transfer,
eqn (1), peptides subjected to ExD reactions must be multiply
charged, as neutral fragments cannot be detected by the mass
spectrometer. Hence, electrospray ionization (ESI) is the technique of choice for ExD MS, since it efficiently generates multiply
charged peptide and protein ions, in contrast to most other
common ionization techniques, e.g., matrix assisted laser
desorption ionization (MALDI). Typically, in acidic conditions,
positive charges are located on amino acids with basic side-chains
(lysine, arginine and histidine), and on the amine group of
the N-terminus. Modifications, such as N-terminal acetylation,
remove a potential charge site. Generally, the peptides are protonated, although use of metal cations or synthetic charge tags can
provide additional charge sites by chemical modifications.11–13
An additional increase in peptide charge, or supercharging, is
achieved by modifying solvent composition and ion source parameters, as discussed elsewhere.14 For simplicity, here only protonation as a means of peptide or protein charging is considered.
2.2. Peptide fragmentation pathways in ExD mass
spectrometry
Scheme 1 illustrates major and minor fragmentation pathways
in ExD MS/MS of a multiply charged peptide cation.2,4 The
major fragmentation channel products result from cleavage of
N–Ca bonds, producing c- and z-ions. The mass of two complementary fragments upon single charge neutralization is
equal to [M + (i + j + 1)H], where M is the mass of the neutral
precursor peptide molecule, while i and j are the charge states
of the c and z ions, respectively. Scheme 1 (major fragmentation
channel products) illustrates the c-ion containing only one
(c1 ion) or n 1 (cn1 ion) amino acids, as well as z-ions
containing only one (z1 ion) or n 1 amino acids (zn1 ion),
with n being the total number of amino acids in the peptide.
More rarely, and at lower abundances, a and y ions may be
observed in the ExD mass spectra, see Scheme 1 (minor fragmentation channel products) for a1 , an1 , y1, and yn1 ion
structures. These fragments are believed to arise when the
charge is solvated not on the amide oxygen, but on the backbone
amide nitrogen atom. Because they occur infrequently, these
fragments are rarely used for sequencing. Note that for the z1 or
y1 fragment to be charged, the amino acid side-chain must be
protonated, which, in practice, limits their observation to fragments containing lysine, arginine, and histidine amino acids.
ExD fragment ions may decompose as a result of neutral
losses such as water and ammonia, or losses of parts of or
entire side-chains. The side-chain losses can be amino-acid
specific or may come from a small subset of amino acids,
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Fig. 2 Illustration of radical/even-electron pairs for c and z ions, commonly
found in ExD MS/MS of peptides. Both top and bottom panels may correspond to
mixtures of either z and z 0 or c and c 0 species.
Scheme 1
Major and minor products of ExD MS/MS.
providing an additional way to confirm the primary structure
(sequence).15 Additionally, it is possible to observe species
where electron capture results in a single hydrogen atom loss.
The signals from these species will overlap with the isotopic
distributions of the parent ions, as shown in Section 4,
vide infra. Note that for multiply charged species more than
one electron capture event may occur, therefore more than one
hydrogen atom loss may be observed.
A special facet of the c/z ion formation is that, oftentimes, it
is possible to observe a radical c-ion and an even-electron z-ion,
in addition to c 0 and z ions.16 It is postulated that after the
N–Ca bond cleavage of [M + nH]n+, the [c + z](n1)+ complex is held
together by non-covalent interactions, before breaking apart.17–20
If the lifetime of this complex is sufficiently long, the z radical
fragment may abstract a hydrogen atom from the c 0 fragment,
leading to formation of z 0 (z 0 = z + H) and c (c = c 0 H) species.
Hydrogen abstraction occurs to varying degrees in different complexes, which leads to a signal in the MS/MS spectrum that is a
combination of the two isotopic distributions of the radical and
even-electron components of a particular ion, Fig. 2.
The difference between the two left-most peaks in both
isotopic clusters in Fig. 2 corresponds to the mass of a hydrogen
atom (B1.0078 Da), whereas the difference between the two
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right-most peaks equals B1.0033 Da, which corresponds to the
difference between the 12C and 13C isotopes. This increases the
complexity of the MS/MS spectra, since from the composite
isotopic distribution it is not readily apparent whether a fragment is a z-radical ion scavenging a hydrogen atom, or a c-ion
losing a hydrogen atom. In both cases, the left-most peak in the
isotopic distribution is the monoisotopic peak of the radical
species, and the peak directly to the right of it may contain the
monoisotopic peak of the even-electron species.
Several methods have been developed to differentiate c and z
ions in ExD mass spectra. One, reported by Coon et al.,21 expands
upon the ideas underpinning the nitrogen rule, noting that oddelectron z-ions are composed of an even number of odd-valence
atoms (e.g., N), whilst the even-electron c-ions, along with b and
y ions, have an odd number of odd-valence atoms in their
elemental compositions. Although this method has been shown
to work in large-scale proteomics studies, there are two main
drawbacks: (i) the accurate mass of fragment ions giving unique
elemental compositions must be known; (ii) given an exact
elemental composition, a species can be determined to be a
radical, but isotopic clusters of c-ions and z-ions may be very
similar, and the method is unable to distinguish c and z species.
A second method of distinguishing N-terminal and C-terminal
fragment ions, reported by Tsybin et al. for ECD MS/MS,19
operates on the principle that a c , z 0 pair is formed if the
[c + z](n 1)+ complex has a relatively long lifetime before
separation, which allows time for a hydrogen transfer from the
c 0 fragment to the z fragment to occur. If the internal energy of
the precursor ion is increased (e.g., using ion activation by
photons from IR laser as employed in activated ion-ECD or
directly with more energetic electrons in ECD), the [c + z](n 1)+
complex will have a shorter lifetime that reduces the probability
of H-transfer and suppresses c , z 0 pair formation. Experimentally, ion activation in conjunction with ECD will affect the
relative abundances of the resultant isotopic distribution by
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varying the ratio of radical/prime species contributing to the
mass spectral signal. Therefore, it could be possible to distinguish the c and z ions, with the drawback being that two mass
spectra, both with and without ion activation, must be obtained
for each precursor peptide in order to observe isotopic patterns.
Another limitation is that the method requires a radical component of c-ions or an even-electron component of z-ions to be
present in the ECD mass spectrum, which is not always the case.
In contrast to low-charge state peptide ion fragmentation,
ExD of protein ions yields primarily even-electron c-ions and
radical z-ions. Presumably, Coulombic repulsion between complementary protein fragments with higher charge states
reduces the lifetime of the intermediate [c + z] complex.17,19
Reducing the precursor protein charge state may enhance the
yield of radical c-ions and even-electron z-ions. For example,
ECD of ubiquitin, a protein of B8.6 kDa, in charge states below
+7 produces abundant radical c6 ions, which disappear when
the protein charge state increases above +7. Similar observations have been reported for ETD of peptides.7
2.3.
Other electron–ion/ion–ion fragmentation methods
Interaction of more energetic, B10 eV, electrons with multiply
charged peptide cations, known as hot ECD (HECD), results in
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increased efficiency of secondary fragmentation, leading to
extensive amino acid-specific side-chain losses (e.g., formation
of w-ions).2,4 Electrons of even higher energy, up to 20–100 eV,
may induce multiple ionization (by desorption of one or more
electrons) of peptide and protein cations; this is accompanied
by extensive backbone fragmentation where all bonds can be
cleaved, as employed in electron ionization dissociation (EID)
(eqn (2)).23
e desorption
½M þ nHnþ þe ð20 100 eVÞ ƒƒƒƒƒƒ!
Chem. Soc. Rev., 2013, 42, 5014--5030
(2)
ðnþ1Þþ bond cleavage
½M þ nH
ƒƒƒƒƒƒ! fragments
Cleavage of the N–Ca bond is also possible for multiply charged
peptide and protein anions following capture of a slightly more
energetic, B3–6 eV, electron, as employed in negative ion ECD
(niECD) (eqn (3)).24
e capture
½M nHn þe ð3 6 eVÞ ƒƒƒƒƒ!
(3)
bond cleavage
½M nHðnþ1Þ ƒƒƒƒƒƒ! fragments
ETD vs. ECD MS/MS
Although the mechanisms for fragmentation of peptides upon
electron capture or transfer are similar (see below) as a result of
both fundamental and experimental considerations, the ETD and
ECD mass spectra may be quite different. Generally, the energy
imparted into a molecule by the free electron capture in ECD is
greater than during electron transfer in ETD (electron affinity of
the radical anion and the electronic state from which the electron
comes from each play a role). As a result, for very short peptides
(circa 5–6 amino acids), the ECD mass spectrum may show
additional peaks, via the opening of new competitive fragmentation channels. In the most common implementation of ECD on
FT-ICR MS, it is possible to define the average electron energy by
varying the potential difference between the electron emitting
surface (cathode) and the ion–electron interaction region in the
ICR cell, Fig. 3, top panel.22 Nevertheless, to produce sufficient
flux of electrons for efficient ECD MS/MS it is practically very
challenging to reduce electron energy below 1 eV.
From an experimental point of view, the conditions are
significantly different between ECD and ETD. ECD takes place
under high vacuum (B1010 Torr), whereas the ion–ion reactions of ETD take place in ion traps, where the vacuum is
several orders of magnitude lower. The latter condition results
in faster collisional cooling, reducing the internal energy of the
post-electron attachment complex and likely the occurrence of
more energetic fragmentation routes. The initial conformations
and internal energies of the precursor ions, and the lifetimes of
[c + z](n1)+ complexes after N–Ca bond cleavage might also be
affected. The difference in pressure potentially influences the
radical/prime ratios of fragment ions, the relative abundance
of the charge-reduced species and the number of peaks
attributable to neutral losses, as detailed below.
2.4.
Tutorial Review
Ca–C backbone bond cleavage is a minor fragmentation channel
in ExD of peptide cations, but is a preferential fragmentation
pathway for multiply charged peptide anions interacting with
energetic, >10 eV, electrons in electron detachment dissociation
(EDD)2 and its ion–ion reaction analogue, negative ETD (nETD),
where electron transfer from the peptide precursor ion to the
reagent radical cation occurs, Fig. 1, eqn (4).25
e desorption
½M nHn þe ð10 20 eVÞ ƒƒƒƒƒƒ!
(4)
bond cleavage
½M nHðn1Þ ƒƒƒƒƒƒ! fragments
Metastable atom-activated dissociation (MAD) is the only currently known fragmentation technique that leads to cleavage of
all possible peptide backbone bonds and is equally applicable
to singly charged precursor ions in both positive and negative
ion modes.26
2.5.
ExD MS/MS: practical considerations
The efficiency of ECD and associated techniques based on ion
interaction with free electrons, as described above, is a function
of electron energy and the number of electrons. The crosssection of the electron capture by a multiply charged peptide
cation is largest at low electron energies and decreases exponentially with increasing electron energy, becoming insignificant at a few eV.2 On the other hand, the number of electrons
participating in ECD interactions may be increased significantly for high (>5 eV) energy electrons due to their more
efficient extraction from the near cathode region. To provide
a large number of electrons and an optimum overlap of ion and
electron beams, a large electron-emitting surface with an
indirectly heated dispenser cathode (with a diameter of
1–10 mm) is most often employed as an electron source in
modern commercial and home-built instruments. The benefits
and limitations of using dispenser cathodes in ECD MS/MS
have been discussed elsewhere.22 The efficiency of ExD is
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Fig. 3 Schematic diagram of main ExD MS/MS implementations: (top) dispenser cathode-based ion–electron interactions implementation for ECD in FT-ICR MS allowing
simultaneous IRMPD (use of photon beams) and ECD (use of electron beams) MS/MS and (bottom) ion–ion reactions implementation for ETD in (linear, Paul, etc.) an ion
trap mass analyzer.9,22 Reagent radical anions can be produced in the chemical ionization (CI) sources and injected into the ion–ion interaction volume from the front-end
(design by Bruker Daltonics, azulene and fluoranthene are typically employed as reagent molecules) and back-end (supported by Thermo Scientific, fluoranthene is
typically employed). Also, glow discharge (GD) can be employed to form reagent anions from nitrosobenzene with front-end anion injection (supported by Waters).
determined from the analysis of the mass spectra before and
after the fragmentation reaction (eqn (5)):
ExD MS=MS Efficiency
¼
Total number ðabundanceÞ of fragment ions
Total number ðabundanceÞ of precursor ions
(5)
Typically, ECD efficiencies of up to 20% can be achieved for
doubly charged peptide cations, whereas higher efficiencies,
B80–100%, have been reported for highly charged peptide and
protein cations.2 The optimum ion–electron interaction period
(or the electron irradiation period) for ECD of peptide dications is
B50–70 ms and B5–20 ms for higher charged protein cations.4,22,27
These results correlate with early ECD mechanistic studies showing
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electron capture cross section to scale quadratically with precursor
ion charge.2 Ion–ion reaction periods in ETD are comparable with
ECD reaction periods, with typical values of B50–200 ms required
for efficient ETD of peptide dications and 5–40 ms for protein
fragmentation.7,10,28 Dissociation of negatively charged peptides in
EDD and niECD typically requires longer ion–electron interaction
periods, up to seconds.2,24 Ion–ion interaction periods in nETD are
comparable to those in ETD.25 Significantly increased electron
energy in EID, up to 100 eV, causes extensive ionization and
secondary dissociation at long ion–electron interaction periods.
EID, therefore, provides most of the analytically useful information
at very short, 0.1–2 ms, electron irradiation periods.23
In comparison to CID, the efficiency of ExD is lower and,
as a result, CID remains the method of choice for large-scale
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Fig. 4 Peptide fragmentation by complementary MS/MS techniques: (top) isolation of precursor ions (e.g., substance P [M + 2H]2+) followed by (bottom left) slowheating vibrational activation (CID, IRMPD) or (bottom right) electron-mediated ion dissociation (ExD). A peptide may carry a post-translational modification (PTM),
e.g., methionine oxidation, shown with a flag. Note, methionine oxidation is not a labile modification.
MS applications.29 Nevertheless, the wealth of information
provided by ExD MS/MS, complementary to CID MS/MS,
ensures continued interest in method and technique development for ExD. Fig. 4 shows a comparative application of slow
heating methods of fragmentation, CID/IRMPD, and electronmediated ExD, to a doubly protonated neuropeptide substance
P, a reference peptide for ExD characterization.
The results in Fig. 4 demonstrate that, as expected, CID/
IRMPD and ExD provide different ion types.30 Note that ExD
does not usually cleave N-terminally to proline (missing c3
fragment ion in ECD, Fig. 4). This phenomenon is known as
the ‘‘proline effect’’ and results from the cyclic secondary
amine structure of this amino acid. Thus, even if the N–Ca
bond is cleaved, the fragments remain covalently bonded via
the proline side-chain, see Fig. 4 inset. In contrast, CID cleavage
of the peptide (C–N) bond to the N-terminus of proline proceeds efficiently. ExD-induced cleavage of this peptide (C–N)
bond is also sometimes possible. Based on a statistical analysis
of cleavage preferences for 15 000 MS/MS spectra of peptides,
Zubarev and co-workers established that CID and ExD are truly
complementary methods of fragmentation.30 Therefore, the
combined MS/MS data from CID and ExD experiments can
provide more structural information.29
Furthermore, while ExD leads to cleavage of strong bonds, the
weaker bonds are often preserved. Importantly, ExD was shown to
preserve labile bonds, specifically, those between post-translational
modifications (PTMs) and amino acid side-chains, to a substantially greater extent than CID/IRMPD.8 Similarly, ExD was shown to
be a powerful method for studying non-covalent complexes.31
Finally, the ability of ExD to preserve non-covalent interactions is
believed to be sufficient to probe the hydrogen bonds participating
in secondary and tertiary structures of peptides and proteins.32–34
For example, ECD MS/MS distinguishes stereoisomers, or peptides
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with L-amino acids substituted for D-amino acids.35 Furthermore,
sequence-specific relative fragment ion abundance distribution in
ExD MS/MS indicates regions of alpha-helix formation in peptides
and proteins.32,36 Two different theories have been put forth to
explain these observations. It was postulated that ExD is a nonergodic process, i.e. the energy released upon electron capture and
hydrogen atom recombination is randomized over the degrees
of freedom after backbone cleavage.2 Alternatively, N–Ca bond
strength could be reduced once a radical intermediate species
is formed, with the consequent bond cleavage following an
ergodic pathway.2
Structural studies have triggered interest in developing a
predictive model for ExD fragment ion abundance based on
peptide and protein sequence information. Zhang developed an
B200 parameter empirical model based on a statistical analysis
of a large database of ECD and ETD mass spectra of peptides.37
Zubarev et al. reported correlation of ECD cleavage frequencies
with dynamics of hydrogen bond formation to a specific amino
acid in a small protein, Trp cage.38 Tsybin et al. observed a high
correlation of ECD relative fragment ion abundance distribution
with amino acid physico-chemical properties, e.g., hydrophobicity and polarity, for a number of peptides.36 Nevertheless, the
low-predictability of these models limits their routine application in large-scale peptide and protein identification studies.
One of the reasons being dependence of fragment ion
abundances on precursor ion internal energy. Indeed, peptide
and protein ion activation with infrared photons in the
gas phase prior to ExD significantly influences fragmentation
patterns.19,20,34 This ion activation prior to ExD is believed to
disrupt non-covalent interactions in peptides and proteins that
unfolds their structures and facilitates fragment ion separation and electron access to buried regions of a protein. Additionally, increased precursor ion internal energy assists proton
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migration along the peptide backbone, providing recombination energy deposition in different locations.
3. Fundamental aspects of electron capture
and transfer dissociation
3.1.
Cornell mechanism
Scheme 2 illustrates the key steps of the Cornell mechanism for
ExD, as originally proposed by McLafferty and co-workers from
Cornell University (NY).5 Electron attachment occurs at a positively
charged site, typically located on the protonated amine group of the
N-terminus or on a basic amino acid (K, R, H) side-chain, forming a
hypervalent radical N-species (assumed to be caught in a Rydberg
orbital). The captured electron reaches the ground state through
relaxation processes, prompting a hydrogen atom transfer to the
amide oxygen and the formation of a carbon-centred aminoketyl
radical intermediate (panels 3 in Scheme 2 top and Scheme 2
bottom). The backbone N–Ca bond located to the right-side of the
carbonyl group is then cleaved, yielding experimentally observed c0
and z fragments. The resulting charge on the c- and z-fragments
depends on the location of charges on the precursor peptide, as
seen by comparing the top and bottom panels of Scheme 2.
Although the Cornell mechanism adequately explains fragment types consistent with those seen in ExD mass spectra,
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soon after its proposal, experimental evidence of charge-remote
fragmentation in ECD MS/MS was noted. This prompted
several alternative proposals, including the Utah–Washington
mechanism.
3.2.
Utah–Washington mechanism
Originally suggested by Simons and co-workers from the
University of Utah (UT)39 and extended by Turecek and co-workers
from the University of Washington (WA),40,41 this mechanism
proposes direct electron capture into the p* antibonding orbital
of the amide group, forming an aminoketyl radical anion, see
Scheme 3. This is followed by anion neutralization by proton
transfer from a charged site (since the anion is very basic) and
cleavage of the N–Ca bond. The ordering of these steps differentiates the mechanisms (with Washington supposing proton
transfer followed by bond cleavage and Utah the reverse). Note
that the endothermic direct electron capture requires Coulomb
stabilization from remote charge sites as illustrated by Simons
et al.42
Simons et al. also considered electron capture by a positively
charged site, as is in the Cornell mechanism, but rather than
relaxation from a Rydberg orbital into the ground state resulting
in H transfer, they suggested electron transfer onto an amide p*
antibonding orbital as the electron relaxes into lower Rydberg states,
Scheme 2 Cornell mechanism for N–Ca bond cleavage in ExD of peptides and proteins with charge solvation from (top) a C-terminal donor amine group; (bottom)
an N-terminal donor amine group. Note that in both cases c- and z-ions, if observed, are identical in mass.
Scheme 3
The Utah–Washington mechanism for ExD.40
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i.e. before H transfer is initiated.42 Charge-remote cleavages are
explained by the large (>20 Å) Rydberg orbital radii, which
overlap with distant amide groups.
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3.3.
Other selected ExD mechanisms
Zubarev et al. proposed a charge-remote modification of the
Cornell mechanism, wherein the electron is captured by an
amide hydrogen H-bonded to a carbonyl oxygen of another
amino acid.38 The hydrogen then transfers to the carbonyl
oxygen, resulting in the aminoketyl radical and leaving an
anionic amide nitrogen. The charge is then neutralized by
proton transfer from a charged site to the amide group.
O’Connor et al. initially proposed a radical cascade mechanism to explain fragmentation patterns in ECD MS/MS of cyclic
peptides, which implied multiple consecutive fragmentations
arising from a single electron capture event.43 The mechanism
also involves an aminoketyl radical intermediate, where, after
N–Ca bond cleavage, a radical cyclization occurs. This process
results in secondary N–Ca bond cleavage with loss of a neutral
fragment or, alternatively, the C-centred radical abstracts a
hydrogen atom from an a-carbon, propagating the a-carbon
radical along the backbone.
Julian et al. suggested that hydrogen deficient radicals may
be responsible for the observed side-chain losses, along with
small molecule (NH3, H2O) losses, and also contribute to
S–S bond cleavage.44 Specifically, such radicals may be formed
either by direct electron capture at charged sites, or via conversion of hydrogen abundant to hydrogen deficient radicals, as is
the case for the formation of the aminoketyl radical intermediate in the Cornell mechanism.
Tsybin et al. recently suggested the ‘‘enol’’ mechanism,
which differs in two key ways from the Cornell and Utah–
Washington proposals, Scheme 4.45 First, the N–Ca bond is
cleaved to the left (N-terminal side) of the aminoketyl radical,
as opposed to the right (C-terminal side). Second, the N–Ca
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bond is cleaved heterolytically, rather than homolytically
(as proposed by earlier mechanisms). Overall, the mechanism
parallels the Cornell mechanism, up to the formation of the
aminoketyl radical. The N–Ca bond located to the left is then
heterolytically cleaved, with a proton back-transfer from the
carbonyl oxygen to the H donor group. This forms a z ion and a
highly basic zwitterionic c-fragment that frequently scavenges a
proton from the z fragment to form a c 0 ion.
4. Analysis of ECD/ETD mass spectra of
peptides
4.1.
Protocol for peptide sequencing by ExD MS/MS
Peptide structure representation for sequencing with ExD is
shown in Scheme 5. Table 1 provides a list of relevant masses,
including those of side-chains and common chemical elements,
along with equations giving structural compositions of z and c
ions, and their relation to b and y ions. An important feature of
peptide and protein structure is the presence of modifications
found in biological systems. These can be present on peptide
termini, single amino acid side-chains, and as covalent
bonds between side-chains. Examples include amidation of
the C-terminus (mass difference of 0.984 Da due to replacement
Scheme 5 Generic peptide structure representation for ExD MS/MS. The
N-terminal amino acid is indicated in blue, the C-terminal in red. The internal
m amino acids are noted in orange. The side-chains, R, are given in green. Table 1
summarizes the masses of amino acid residues and R groups.
Scheme 4 The ‘‘enol’’ mechanism of ExD; note the same resultant fragment elemental compositions and charges irrespective of hydrogen donor site location
(top panel vs. bottom panel).
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Table 1 Accurate masses of amino acid residues, side-chains, and commonly
found elements and groups present in ExD MS/MS of peptides and proteins.
Colour coding: Scheme 5
(continued)
Fragment ions
Ion type Ion mass
Amino
acid
residues
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Table 1
Ion mass (detailed)
zm+1n+
þ
c0mþ1 n
Gly (G)
57.021464
1.007825
Ala (A)
71.037114
15.023475
Ser (S)
87.032028
31.018390
Pro (P)
97.052764
41.039125
Val (V)
99.068414
43.054775
Thr (T)
101.047678
45.034040
Cys (C)
103.009185
46.995546
Ile (I)
113.084064
57.070425
Leu (L)
113.084064
57.070425
Asn (N)
114.042927
58.029289
Asp (D)
115.026943
59.013304
Gln (Q)
128.058578
72.044939
Lys (K)
128.094963
72.081324
Glu (E)
129.042593
73.028954
Met (M)
131.040485
75.026846
His (H)
137.058912
81.045273
Phe (F)
147.068414
91.054775
Arg (R)
156.101111
100.087472
Tyr (Y)
163.063329
107.049690
Trp (W)
186.079313
130.065674
Fragment relations
z+ + NH2 = y+
Phosphorylation
pXxx (pX)
X = S, T, Y
Proline effect
XxxPro (XP)
PHO3
79.966331
X = any
amino acid
Nomenclature
56.013639
73.040188
58.005479
57.021464
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c+ = b+ + NH3
Elements
Mass (Da)
Elements, groups
Mass (Da)
1
1.007825
12.000000
14.003074
15.994915
31.972071
30.973762
e (electron)
H+ (proton)
NH–O
NH3
13
C–12C
34 32
S– S
0.000549
1.007276
0.984016
17.026549
1.003355
1.995796
H
12
C
14
N
16
O
32
S
31
P
of the OH group by NH2), oxidation of the methionine sidechain (formation of a [M + Ou + nH]n+ species, where u = 1 or 2),
phosphorylation (PO3H, 79.966 Da) of hydroxyl containing sidechains (Ser, Thr, and Tyr), or formation of a disulphide bridge
between two cysteine residues.
To best illustrate the workflow of peptide de novo sequencing, we assume that the MS/MS spectra have been acquired
using a high resolution, high mass accuracy mass spectrometer, e.g., FT-ICR MS, Orbitrap FTMS, or a high performance
TOF MS. The following five steps should be followed for ExDbased peptide sequencing:
(1) Identification of the precursor ion and charge-reduced
species. Since the m/z value of the precursor ion is known,
use the formula q = n 1, where q is the charge state and n is
the number of isotopic peaks per 1 m/z unit, to determine
the charge state of the precursor ion (since 12C and 13C isotopes differ by B1 Da, Table 1). Knowing the m/z value and the
charge state of the precursor ion, it is possible to derive the
m/z value of the charge-reduced species. A multiply charged
precursor ion may capture more than one electron, leading to
[M + nH](nk)+ species, where k is the number of captured
electrons. If k is odd, a radical cation is formed, while even
values of k lead to even-electron species. If hydrogen loss also
occurs, one may observe [M + (n r)H](nk)+ (r r k) species as
well. Since not every electron capture or transfer event leads to
backbone cleavage and fragment ion separation, the reduced
molecular ions are often the most abundant peaks in the
MS/MS spectrum.
(2) Determination of c01 and z1 ions in the lower m/z region.
For tryptic peptides generated in bottom-up experiments (see
below), the most common z1 ions are arginine (159.1002 Da)
and lysine (131.0941 Da). Identification of the z1 ion facilitates
identification of subsequent amino acids.
(3) If the lower m/z region of the MS/MS spectrum is missing,
analysis should start by identification of the complementary
c0n1 and zn1 ions, Scheme 1. In this case one must subtract the
mass values of c0n1 and zn1 from the charge reduced species.
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These yield a mass difference, Mx, equivalent to a neutral c01 or
z1 fragment, see Table 1 for appropriate calculations (Mx C2H5N2O = R or Mx C2H2O2 = R). Given singly charged
fragments, complementary c 0 + z = [M + 2H]+ . Thus, subtracting the mass of one fragment provides the mass of the
complementary ion. Note that the termini might be chemically
modified. Ion signals less than 50 Da lighter than the precursor
ion arise from neutral losses from the precursor ion.
(4) Establishment of a series of c- and z-ions, focusing
primarily on the most abundant peaks. If there is a gap of
>186 Da between two fragment ion peaks, it is advisable to
search for low intensity peaks, as those might indicate an
unfavourable N–Ca cleavage. Proceed by considering the relationship between the masses of consecutive ions of the same
type and compare the masses to the C2H2ON + R values
calculated from Table 1. Ideally, both c- and z-ion series are
present. Then, it should be possible to link up either of the
sequences to a c1 or z1 fragment ion, or by taking the difference
between the largest identified ion in either sequence and the
charge-reduced species, e.g., [M + nH]+ . For the peaks that
appear to be a radical/prime isotopic distribution, it is useful to
note the two leftmost peaks, as either could be a c 0 or a z ion,
see Fig. 2. In case the c and z ion isotopic distributions overlap,
use the Coon et al. or Tsybin et al. methods described
above.19,21
(5) If the N–Ca bond between a pair of amino acids is not
cleaved, or an ion series is missing, cross-check with the
complementary c or z ion series to complete the gaps. Consider
the possibility of gaps due to the proline effect, combinations of
the masses of two amino acids and that of specific neutral
losses, Table 1. Check for the presence of a 34S isotope in
fragments above and below the gap. Consider MS/MS spectra
from other fragmentation techniques, e.g., CID, that may
include cleavages between missing amino acid pairs.
4.2.
Case study: ExD of an unmodified model peptide
Fig. 5 shows a comparison of ECD and ETD mass spectra of an
isolated multiply charged peptide cation located at 391.177 m/z.
The first step is to identify the charge of the precursor
species, as per protocol point 1 above. In this case, there are
four peaks per m/z unit (data not shown), hence the ion in
question is triply charged. Since ESI was used, the species is
assumed to be triply protonated, i.e., the parent species is
[M + 3H]3+. The charge-reduced species are then doubly
charged, [M + 3H]2+, and should be located at 586.770 m/z,
and the singly charged species, [M + 3H]+, located at
1173.543 m/z. Such species are, indeed, found, and their charge
states confirmed, Fig. 5 insets. Additionally, Fig. 5 shows the
presence of multiple species, doubly charged in the mid-range,
and singly charged at the high mass end of the MS/MS spectrum.
Species found in the 1050–1175 m/z region include the [M + 3H]+
ion, found at 1173.54 m/z, with [M + 2H]+ and [M + H]+ species
contributing to the observed isotopic distribution, and combinations of various neutral losses resulting in ion signals found at
lower m/z values, see Fig. 5 inset and bottom panel, respectively.
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Fig. 5 Electron-mediated fragmentation of a triply protonated peptide NEREEHAMR–OH: (top) ECD FT-ICR MS with low energy, B1 eV electrons and 70 ms
ion–electron interaction period; (bottom) ETD LTQ Orbitrap FTMS with a 100 ms
interaction period with fluoranthene radical anions. z7 and z8 ions are present in
the ETD mass spectrum only.
A typical z1 ion of Arg at 159.100 m/z is found in the low m/z
region (see Tables 1 and 2, Schemes 1 and 5), Fig. 6. With the
z1 peak identified, the z-ion series can be deduced. As can be
seen from Fig. 5 and 6 (see Table 2 for theoretical and
experimental mass values of singly charged fragment ions), it
is possible to follow the z-ion series all the way up to the
1041.466 m/z peak (value also confirmed with a doubly charged
ion in the MS/MS spectrum at B521 m/z).
If the second peak in the isotopic envelope of a singly
charged ion is B1.008 m/z away (see Fig. 6 insets), it indicates
the presence of multiple contributing ion species, typically
representing a radical and an even-electron ion pair (see
Fig. 2). This is illustrated by the ion at 1042.470 m/z: there is
a small radical species peak at 1041.463 m/z, 1.008 m/z lighter,
clearly pointing to a radical–prime pair. In these cases, both
peaks must be noted, as either can be part of a c- or z-ion series.
Here, it is possible to choose the correct peak: if it is part of the
z-ion series, the difference between any ion of the same type
should yield an even-electron mass-fragment corresponding to
one of the amino-acid masses. Otherwise, refer to Coon et al.
and Tsybin et al. methods to identify the ion type, as per
protocol point 4.
Finally, the remaining gap to the molecular ion from the
z-ion with highest mass is the N-terminal amino acid. The
difference between the two peaks is 131.0696 Da; accounting for
the N-terminus, 131.0696 C2H5N2O = R = 58.0293 Da = RAsn
(Table 1). Therefore, the sequence reads: H–NEREEHAMR–OH.
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Table 2 Mass values for ExD MS/MS singly protonated fragment ions of model
peptides analysed in Fig. 5–7: theoretical (top value in each cell) and experimental (shown in bold, bottom value in each cell). Experimental values are shown
corresponding to a routine mass accuracy level and may be further corrected by
re-calibration. N/A – ion not found
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H–NEREEHAMR–OH
0þ
ctheor
0
cexp þ
ytheor
yexp+
+
H–SWApTCH–NH2
0
ztheor+
zexp+
132.0768
175.119
159.1002
N/A
175.1190 159.1004
261.1193
306.1594 290.1407
261.1188
306.1587 290.1401
417.2205
377.1966 361.1778
417.2197
377.1953 361.1772
546.2631
514.2555 498.2367
546.2614
514.2556 498.2351
675.3056
643.2981 627.2793
675.3032
643.2950 627.2771
812.3646
772.3406 756.3219
812.3621
772.3375 756.3192
883.4017
928.4418 912.423
883.3986
928.4390 912.4206
1014.4422 1057.4844 1041.4656
1014.4398 1057.4791 1041.4625
ctheor þ
0
cexp þ
1 105.0659
N/A
2 291.1452
N/A
3 362.1823
N/A
4 543.1963
543.1956
5 646.2055
646.2047
6
ytheor+
yexp+
ztheor+
zexp+
155.0927
155.0926
258.1019
258.1016
439.1159
439.1154
510.153
510.1526
696.2324
696.2316
139.074
139.0738
242.0832
N/A
423.0972
423.0967
494.1343
494.1337
680.2136
680.2128
7
8
Fig. 6 Expanded m/z segments of ETD LTQ Orbitrap FTMS mass spectrum of
triply protonated peptide NEREEHAMR–OH (see Fig. 5). Insets demonstrate
isotopic distribution of several fragment ions.
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Most of this sequence can be verified via the c ions, as well as
with a number of low abundant y and a species, see Fig. 5 and
Table 2. It is worth noting the expanded view of the peaks
identified as z2+ , z4+ and z5+ , where the isotopic fine structure
shows the presence of the 34S isotope, circa 1.995 m/z heavier
than the singly charged monoisotopic peak, confirming the
presence of a sulphur-containing methionine next to the
C-terminus.
Interestingly, the extent of charge-reduced and radical fragment ion formation is significantly different between ECD and
ETD MS/MS, Fig. 5. The radical charge-reduced species are
more abundant in ETD than in ECD ([M + 3H]2+ in ETD vs.
[M + 2H]2+ in ECD), [M + 3H]+ is observed in ETD but not in
ECD, and ETD shows a more pronounced radical z-ion series.
Potential causes for these differences are discussed in
Section 2.
4.3.
Case study: ExD of a model phosphorylated peptide
Fig. 7 shows the ETD mass spectrum of a precursor ion located
at 392.135 m/z. The precursor species is doubly charged. The
charge-reduced ion, [M + 2H]+ , is located at 784.271 m/z, see
Fig. 7 top, right inset. As in the previous example, the chargereduced ion peak is a part of an overlaid isotopic cluster. We
may observe a combination of the charge-reduced species
found at 784.271 m/z and the precursor ion which, upon
electron capture, fragmented via H loss, gives rise to the
[M + H]+ species at 783.263 m/z.
Fig. 7 ETD LTQ Orbitrap FTMS of a doubly protonated phosphopeptide
SWApTCH–NH2: (top) 100–800 m/z MS/MS spectrum and (bottom) 400–700 m/z
expanded segment showing fragment ion isotopic features.
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Next, consider the radical–prime pair at 139.074/140.082 m/z,
Fig. 7 top, left inset. It does not fit the typical z1 ions with R,
K, or H amino acids at the C-terminus. By using Table 1 and
knowing that the fragment is charged but the ion type is
unknown, one can subtract one proton, followed by either the
c1 or z1 backbone part, to obtain the side-chain, R (139.074 H+–C2H5N2O and 139.074 H+–C2H2O2). This procedure can
be repeated for the ion at 140.082 m/z. As can be seen, none of
the R masses fit the remainder. The most common reason is
that the side-chain or the terminal group of the amino acid is
modified, see protocol point 5. One of the most common modifications for a terminal group is amidation of the C-terminus. By
adding the mass of NH and subtracting that of O, Table 1, one can
get the expected z1 fragment from the C-terminus. Considering
this modification, R now matches histidine (His, H) with an
amidated C-terminus.
Attempts to find the next ion in the sequence from this end of
the MS/MS spectrum have been unsuccessful, as none of the peaks
match the mass of the known amino acids. Taking the difference
between 784.2705 m/z and 680.213 m/z peaks, then subtracting the
values for the c1 and amidated z1 backbone parts (73.04019 Da and
57.02146 Da, respectively) and comparing to the R values for
amino acids (see Table 1), serine can be identified (meaning that
680.213 m/z is the zn1 ion). Repeating the procedure for the
646.205 m/z peak identifies this as the cn1 ion, with residue match
for histidine, which confirms the z1 ion assignment, Table 2.
Establishing m/z differences between other ion signals and
comparing them to the values from Table 2 provides the sequence
tags H–SWA. . . and . . .CH–NH2. A careful look at the data shows a
gap in the z ion series of B300 Da between ions at 139.074 m/z
and 423.097 m/z. Since from the c-ion series the identity of the
penultimate C-terminal amino acid is established, one can determine the hypothetical mass of the z2 ion and use this value
(242.0832 Da) to determine the mass of the remaining gap in the
sequence by subtracting it from 423.097 Da, giving 181.0138 Da.
Subtraction of the amino acid backbone mass from 181.0138 Da
gives 125.00016 Da – note that the mass defect for this mass is
rather low. Indeed, if one considers the mass defects for the most
common elements encountered in organic compounds (Table 1),
only sulphur (S) and phosphorus (P) have significant mass defects
which can reduce the mass, countering the effects of at least several
hydrogen atoms present. Therefore, it is likely that the one or two
amino acids in question have at least one S or/and P atom present.
This generally narrows down the scope, as only 2 amino acids
contain sulphur (M and C), and only 3 (S, T, Y) can be modified by
phosphorylation (PHO3 group). By subtracting the side-chains of M
and C, and, separately, by subtracting PO3H from 125.00016 Da, it
can be seen that the remainder for PHO3 subtraction is equivalent
to the mass of a threonine (Thr, T) side-chain. The peptide
sequence established with the z-ion series is: H–SWApTCH–NH2.
Note that the presence of phosphorylation is typically revealed by a
characteristic loss (B80 Da) from the precursor ion in CID/IRMPD.
Finally, as can be seen from Table 2, a complete y-ion series
is, indeed, present in the MS/MS spectrum, albeit most peaks
are of low intensity, and are likely to be missed during the
initial most-abundant peak assignment. Additionally, the high
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m/z region of the MS/MS spectrum contains numerous peaks
arising from neutral side-chain losses which, upon closer
examination, include loss of phosphorylation, as well as partial
and complete losses of side-chains from several amino acids in
the sequence. This information may then be used to further
confirm the peptide sequence.15
5. Selected applications of ECD/ETD MS in
the life science research
5.1. Proteolysis-based proteomics: bottom-up and
middle-down approaches
Bottom-up and middle-down are the major proteolysis-based
approaches of MS-based proteomics.8,46 For bottom-up proteomics, the most commonly employed protease is trypsin, which
cleaves at the C-terminus of basic amino acid residues, namely
Arg (R) and Lys (K).4 The resulting tryptic peptides are on average
10 residues-long and, consequently, their maximum charge state
is rarely higher than 3+, reducing de facto the efficiency of the
ExD process.7 The activation time for low charge state peptide
ions remains high (up to 100–200 ms), thus the MS/MS analysis
by ExD is not ideal for the high sample complexity and time
constraints typical for the bottom-up approach, for which CID
and HCD (higher-energy collision dissociation)47 are the most
frequently used ion activation and dissociation techniques. ExD
remains the method of choice for localization of labile PTMs,
however, the long activation time and high number of precursor
ions required to yield abundant fragment ions significantly
decreases the duty cycle.8
A possible solution to exploit the advantages of ExD for
proteomics is the employment of enzymes with alternative
cleavage specificities.46 Generation of longer peptides leads to
overall higher charge states and results in a more efficient and
faster ExD process. In addition, the smaller number of peptides
generated from the same protein mixture results in reduced
sample complexity. This approach, known as middle-down
proteomics, has first been attempted by limited proteolysis27
and acid hydrolysis.48 A number of proteases, including LysC,
LysN and AspN, have been shown to provide longer average
peptides than trypsin. Recently, novel proteases that yield even
longer peptides, therefore greatly increasing the average charges,
have been proposed. Specifically, OmpT (by the group of Kelleher) and Sap9 (by the group of Tsybin) are the two proteases
recently employed in middle-down proteomics that cleave at
dibasic sites (i.e., between or before two consecutive basic amino
acids, which can be Arg (R) or Lys (K)).46 The bottom-up and the
middle-down approaches share a common workflow regarding
sample preparation and liquid chromatography (LC) separation.
However, due to the increased length of the peptides in middledown proteomics, the analysis of the fragment ions has to be
performed in a high resolution mass analyzer.
5.2.
Proteolysis-free proteomics: the top-down approach
The term ‘‘top-down mass spectrometry’’ (TD MS) refers to the
mass analysis of intact proteins and large protein fragments
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followed by gas-phase fragmentation and mass analysis of their
fragment ions.49 In contrast to bottom-up and middle-down
approaches, TD MS does not apply any proteolytic digestion of
proteins in solution prior to MS.50 The combination of intact
protein mass measurement in MS and the identification of
product ions generated in the subsequent MS/MS event overcomes some of the traditional problems related to the proteolytic peptide-based strategies. Specifically, TD MS allows the
identification of a single proteoform (or a protein isoform), and
not of a protein family as in the case of bottom-up and middledown MS.50 The high complexity of MS/MS spectra populated
with highly charged fragment ions limits the TD MS approach
to high resolution mass spectrometers, Fig. 8.10,28
At first glance, ECD and ETD of the protein in the same
charge state provide similar fragmentation patterns, Fig. 8.
However, closer examination reveals some differences between
charge-reduced ion distributions, as well as in the c- and z-ion
partitioning. First, ECD may give more charge-reduced species
(up to triply neutralized precursor ions carrying 19 charges, not
assigned here) than ETD, Fig. 8. ECD also produces more
intense neutral losses from all the above-mentioned chargereduced species. Furthermore, z366+ ions are present only in the
ETD MS/MS spectrum, and not in the ECD one, whereas z6+ is
assigned uniquely in the ECD MS/MS spectrum, Fig. 8 left
insets. Finally, in the higher m/z region of the MS/MS spectra
(Fig. 8 right insets) the same product ions show different
relative intensities in ETD vs. ECD (e.g., c172+).
Chem Soc Rev
ECD and ETD are particularly suited for TD MS as they
generally result in a more extended protein fragmentation (and
hence sequence coverage) than slow-heating activation techniques such as CID and IRMPD.2,4 While slow heating methods
cleave the weakest peptide bonds, and guarantee information
limited to the C- and N-terminal ends of proteins (which are
often structurally flexible), ECD and ETD may also cleave the
middle portion of protein sequences as they may deposit energy
near the charged sites.10,34 In addition, the suggested propensity toward cleaving disulphide bridges by ExD might be useful
when analyzing protein or protein complexes in their ‘‘native’’
form, i.e. without reduction and alkylation prior to ionization.31
At a practical level, the multiply-charged protein cations
resulting from ESI are ideal for ECD and ETD, because the
fragmentation efficiency increases substantially with the precursor ion charge state, as previously mentioned. The high
charge state of proteins allows sensible reduction of the
electron irradiation (for ECD) or ion–ion interaction (for ETD)
period when compared to the values typical for peptide
analysis. Characteristic TD MS activation periods can vary from
2–5 ms to B50 ms, depending on the protein’s molecular
weight, selected precursor charge state and instrument. It must
be noted that a prolonged ion–electron or ion–ion interaction
period might induce multiple electron capture and transfer
events, with subsequent formation of internal fragments,
which remain a challenge for identification with any TD MS
analysis software.
Fig. 8 ExD MS/MS of [M + 22H]22+ ions of a B17 kDa protein (horse myoglobin) showing (top) ECD FT-ICR MS, an ion–electron interaction period of 8 ms; and
(bottom) ETD LTQ Orbitrap FTMS, an ion–ion interaction period of 3 ms. The left insets on both panels show the presence of low charge state fragment ions, up to
B700 m/z. The right insets illustrate the complexity of the MS/MS spectra in the high m/z region.
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Besides the activation period reduction, other specific
settings can be tailored for ExD when applied to TD MS. An
increased signal/noise ratio of fragment ions in ExD can be
obtained by enhancing the separation of non-covalently bound
charge-reduced intermediate complexes by precursor ion activation, e.g., with an IR laser prior to ECD or ETD reactions.20,51
When proteins are analyzed by ETD, the target number of
electron carrier molecules used in a single ETD event is also
relevant. Although standard settings employed for peptide
analysis might work well, for large proteins (>50 kDa), which
require extremely short activation periods, the efficiency of
the process is improved by increasing the number of radical
anions.
The signature feature of ETD and ECD of retaining labile
PTMs while fragmenting the peptide backbone has been
exploited also for intact protein analysis. ECD FT-ICR MS was
applied, for instance, for determination of the phosphorylation
profile of mouse cardiac troponin I by Ge and co-workers.52
Impressively, ExD also showed its potential in revealing the
position of single amino acid mutations53 and, recently, in
detecting position (and quantity) of isoAsp residues resulting
from deamidation events.54
Notably, when maximum sequence coverage is needed
during investigation of a selected, isolated protein, recent
results show the importance of searching ExD-generated product ions not only against a list of theoretical c- and z-type ions,
but also against y-type ions, that are frequently generated
during the ExD process.28
5.3. ExD MS in structural biology studies of proteins and
protein complexes
ECD and ETD have been applied recently with success to both
in-solution and gas phase hydrogen–deuterium exchange
(HDX) MS to identify the dynamics of protein deuteration sites
with single amino acid resolution. The main problem of
MS/MS-based HDX is the migration of hydrogen and deuterium
atoms along the polypeptide backbone (a phenomenon known
as ‘‘scrambling’’) during tandem MS. ExD was shown to substantially limit this issue compared to CID,55 especially when
large systems such as intact proteins are investigated. For
smaller systems, e.g., peptides, initial applications of ExD to
HDX MS generated a number of controversial results, indicating a sequence-specific response of HDX rates to ExD.56
Currently, a combination of ExD and HDX MS is applied,
mainly in a top-down approach, to address peptide and protein
structural biology questions (in-solution HDX) or reveal their
gas phase conformations (gas phase HDX). Pulsed labeling
HDX MS has been used by Konermann and co-workers for
obtaining information about the folding dynamics of a small
protein, apo-myoglobin, through ECD top-down MS of its
short-lived folding intermediates.57 Other possible applications
of in-solution HDX MS include the determination of protein
binding sites. Gross and co-workers determined the C-terminal
portion of apolipoprotein E as responsible for its oligomerization and detailed the structural elements involved using an
ETD-based bottom-up MS approach.58 Conversely, ECD top-down
5028
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Tutorial Review
HDX MS was carried out for elucidating the aggregation
dynamics as well as the secondary and tertiary structures of
the aggregated form of the amyloid beta 1–42 peptide.59
With regard to non-labeling experimental strategies, ExD
top-down MS was shown to be powerful for assessing the
structure of non-covalent protein complexes when employed
in combination with native ESI. The latter refers to ESI performed under particularly soft conditions and from protein
solutions close in composition and pH to the physiological
conditions.60 Native ESI MS generates significantly lower and
narrower charge state distributions of macromolecular assemblies. Therefore, protein sequence coverage by ExD in native MS
may be reduced compared to the denaturing ESI MS. Nevertheless, Gross and co-workers have recently determined the
composition and stoichiometry of different protein complexes
by applying ECD to pre-activated protein assembly precursor
ions.31
6. Conclusions
This tutorial provides a brief overview of fundamental, analytical, practical, and applied aspects of ExD-based MS for peptide and protein structure analysis. ExD MS data interpretation
guidelines for peptide de novo sequencing are provided and
applied to step-by-step sequencing of unmodified and phosphorylated model peptides. Fragmentation patterns generated
by ECD and ETD are compared for a model peptide and a
standard protein, demonstrating not only the similarities
between the two techniques, but also notable differences in
the extent of charge-reduced ion formation and partitioning
between c- and z-ions. Herein, we provided a brief description
of selected applications of ExD MS in life science research
that touches areas of bottom-up, middle-down, and top-down
proteomics, as well as structural biology of proteins and protein
complexes.
Acknowledgements
We thank Anton Kozhinov, Kristina Srzentić, Aleksey Vorobyev,
and Hisham Ben Hamidane for discussions and technical
assistance in manuscript preparation. We are grateful for
financial support through the Swiss National Science Foundation (SNF project 200021-125147/1) and the European Research
Council (ERC starting grant 280271).
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