Study of the gas-phase fragmentation behaviour of sulfonated peptides

Abstract

A series of singly and doubly protonated peptides bearing sulfonated residue have been studied, using both experiment and molecular modelling, to elucidate fragmentation chemistry of sulfonated peptides. Collision-induced dissociation mass spectra indicate that the sulfo group loss (neutral loss of 80 Da) is the dominant dissociation channel. Modelling results suggest the proton transfer mechanism, where upon vibrational excitation, the acidic side chain proton is transferred from the sulfo group hydroxyl to the ester oxygen resulting in S-O bond cleavage and formation of the unmodified hydroxyl containing residue and SO3. Conformations associated with potential energy profile of the reaction imply the charge remote nature of the proposed mechanism. The proposed proton transfer mechanism was compared with the intramolecular nucleophilic substitution (SN2) mechanism, the main pathway suggested for neutral loss of phosphoric acid from phosphopeptides. Both pathways (proton transfer and SN2) are available for sulfonated and phosphorylated peptides; however, each posttranslational modification favours different mechanism. The change of the bond dissociation enthalpies and the ability of stabilising the transition state structures are demonstrated as main factors responsible for each posttranslational modification activating a different pathway.

Full text

Study of the gas-phase fragmentation behaviour of sulfonated peptides
Sanja Škulj, Marko Rožman
a b s t r a c t
A series of singly and doubly protonated peptides bearing sulfonated residue have been studied, using both experiment and molecular modelling, to
elucidate fragmentation chemistry of sulfonated peptides. Collision-induced dissociation mass spectra indicate that the sulfo group loss (neutral loss of
80 Da) is the dominant dissociation channel. Modelling results suggest the proton transfer mechanism, where upon vibrational excitation, the acidic side
chain proton is transferred from the sulfo group hydroxyl to the ester oxygen resulting in S O bond cleavage and formation of the unmodified hydroxyl
containing residue and SO3. Conformations associated with potential energy profile of the reaction imply the charge remote nature of the proposed
mechanism. The proposed proton transfer mechanism was compared with the intramolecular nucleophilic substitution (SN2) mechanism, the main
pathway suggested for neutral loss of phosphoric acid from phosphopeptides. Both pathways (proton transfer and SN2) are available for sulfonated and
phosphorylated peptides; however, each posttranslational modification favours different mechanism. The change of the bond dissociation enthalpies
and the ability of stabilising the transition state structures are demonstrated as main factors responsible for each posttranslational modification
activating a different pathway.
1. Introduction
One of the most common uses of mass spectrometry (MS) is
obtaining the structure or sequence of an ion being analysed. The
gas-phase approach to generate structure-specific information
involves use of tandem MS and subsequent interpretation of frag-
ment ion spectra. In the tandem MS of the protonated peptides,
the ion of interest is isolated and then (usually) dissociated via low
energy vibration excitation (either collision-induced dissociation
(CID) or infrared multi-photon dissociation (IRMPD)). Assignment
of molecular structures to the tandem MS spectra greatly relies on
the fragmentation models used. The most comprehensive set of
rules for understanding of dissociation mechanisms of protonated
peptides is known as the mobile proton model [1–5]. The model
(introduced by Vicki Wysocki and Simon Gaskell) assumes that in
the activated protonated peptide, ionising proton(s) can migrate
to various sites, thus triggering charge-directed fragmentation
mechanisms [1–5]. The mobile proton concept has been success-
fully applied in numerous studies of fragmentation mechanisms
of various tryptic and non-tryptic peptides, cyclic peptides and
peptides bearing some posttranslational modification (PTM) [1–7].
Sulfonation, common PTM in multicellular eukaryotes, repre-
sents addition of sulfonic acid group to a protein. Modification
is detected on Tyr (mainly), Ser and Thr residues and has the
same nominal mass increase as phosphorylation (+80 Da) [8–10].
Thus, it was suggested that rates of protein sulfonation could be
underestimated due to a coexistence of both phosphorylated and
sulfonated (isobaric) forms of the same peptide [8–10]. Use of
ultra-high resolution (accurate mass) measurements, ultraviolet
and infrared photodissociation spectroscopy techniques demon-
strated that PTM modified isobaric peptides could be distinguished
[11–13]. Despite the advantages of high-resolution mass measure-
ments, photodissociation techniques and electron capture/transfer
dissociation [14], CID remains the generally used approach for
sulfonation assignments [8–10,15–17]. Protonated sulfonated pep-
tides analysed by CID undergo the facile neutral loss of the sulfur
trioxide (SO3, loss of 80 Da) from their precursor ions [8–10,15–17].
The neutral loss precedes any peptide backbone fragmentation,
thus limiting precise localization of the sulfonation sites. Under-
standing of the gas-phase dissociation pathways of sulfonated
peptides is very limited. Recently, Patrick et al. preformed structural
investigation of protonated sulfoserine dissociation pathways [18].
Low-energy CID of protonated sulfoserine produced two major

product ions: 3-member aziridine ring structure attributed to the
neutral loss of 98 Da (loss of H2SO4) and a structure identical to
the protonated serine was related to the loss of 80 Da (loss of SO3).
However, insights at the peptide level are still missing.
Better understanding of the fragmentation chemistry of sul-
fonated peptides would be of value. Previously, we provided
a description of the gas-phase dissociation of phosphorylated
peptides [7]. One may consider sulfonation and phosphoryla-
tion as similar because both modifications represent highly acidic
monoesters of their respective acids and give rise to a nominal mass
increase of 80 Da However, neutral loss during vibrational exci-
tation from protonated sulfopeptides is associated to SO3[8–10]
while that from phosphopeptides (phosphorylated Ser and Thr
residues) is associated mainly to elimination of H3PO4[6,7], i.e.
suggesting activation of different dissociation pathways.
In this work, through the combination of low-energy CID exper-
iments and molecular modelling, we present the description of the
fragmentation mechanisms of sulfonated peptides. Furthermore,
we complement the present findings on sulfopeptides with our pre-
vious results on phosphopeptides and attempt to understand why
a certain fragmentation pathway is related to specific PTM.
2. Materials and methods
2.1. Materials
Analytes and reagents were obtained from Sigma-Aldrich (St
Louis, USA) and used without further purification. Peptides TSQLL,
SAALSLLR, SAALYLLR and their posttranslational modified variants
were obtained from PolyPeptide Laboratories (Strasbourg, France).
Peptide sulfonation was achieved using the procedure described
in ref. [9]. Briefly, peptides were dissolved in trifluoroacetic acid
and reacted with 5% chlorosulfonic acid (ClSO3H) at room temper-
ature for 20 min The reaction was terminated adding H2O and the
solution neutralised with NH4OH.
2.2. Mass spectrometry
MS and tandem MS experiments were carried out on the amaZon
ion trap mass spectrometer (Bruker Daltonik, Bremen, Germany).
Peptides were dissolved in 50/50 ethanol/water with 0.1% formic
acid to obtain 1 ␮M concentration. Solution was introduced into the
electrospray ionisation source by direct infusion at the flow rate of
75 ␮L/h. The capillary voltage was set at −4500 V while high voltage
end plate offset was −500 V. The temperature and the flow rate
of the drying gas were set at 205 ◦C and 5 L/min, respectively. The
electrospray ionisation source parameters were optimised to allow
an efficient ionisation and to reduce the in-source fragmentation
of precursor ions. The isolation width of the precursor ion was set
at 2 Da The CID excitation time was 40 ms and the amplitude was
in the 0.4–1 V range, depending on a precursor. All spectra were
acquired in the positive ion mode using a scan range from m/z 100
to 1100. DataAnalysis 4.0 and BioTools 3.2 (Bruker Daltonik GmbH,
Bremen, Germany) were used for spectra analysis and extraction of
the MS and tandem MS data.
2.3. Computational methods
In order to gain initial understanding of the potential energy
surface (PES) associated with loss of the sulfate modification, the
proposed pathways were first established on the small model sys-
tem, CH3COsSerNHCH3, and then further evaluated on the test
peptides TsSQLL and SAALsSLLR. Both the small model system
and the peptides were optimised at the B3LYP/6-31G(d) level of
theory. Both the functional and the basis set represent a good
compromise for obtaining satisfactory geometries and approxi-
mate relative energies, as demonstrated in the theoretical studies
of similar systems [7,19,20]. Stationary points (i.e. the minima and
transition states on the potential energy surface) were identified
by the harmonic frequency analysis. Transition state structures
were additionally tested by the Intrinsic Reaction Coordinate (IRC)
analysis. In order to get a more accurate description of dissocia-
tion energies, calculations using the G3(MP2)//B3LYP protocol [21]
were performed on a restricted number of the small model sys-
tem conformations. The G3(MP2)//B3LYP results were correlated
with series of the single point energy calculations in order to pin-
point a model suitable for use on the test peptides. The B2PLYP,
B3LYP, M062X and MP2 methods were used in combination with
the different basis sets: 6-31G(d), 6-31+G(d), 6-31++G(d,p), 6-
311++G(d,p) and TZVP. The smallest mean absolute deviation
was found for energies calculated at the B3LYP/TZVP level of
theory.
Combination of quenched dynamics and simulated annealing
with the AMBER 99 force field was used to sample the poten-
tial energy surface of the test peptides (TSQLL and SAALSLLR) by
the protocol identical to that previously used [7,22]. Final struc-
tures were reoptimised using the B3LYP/6-31G(d) level of theory
and the lowest energy structure was considered as the represen-
tative structure. From the representative structure, the potential
energy profiles of dissociation pathways were constructed at the
B3LYP/TZVP//B3LYP/6-31G(d) level. The Rice-Ramsperger-Kassel-
Marcus (RRKM) kinetic theory was used to describe the reaction
rate as a function of internal energy of peptides.
All quantum mechanic calculations were established using the
Gaussian 09 [23], molecular dynamic simulations were carried out
using the AMBER 12 [24] and RRKM calculations were obtained
using the MassKinetics 1.15 [25].
3. Results and discussion
3.1. CID of sulfonated peptides – mobile proton environment
The low-energy CID product ion spectra were examined in
order to set initial understanding of the gas-phase fragmentation
behaviour of sulfonated peptides. The spectra obtained by dissoci-
ation of the doubly protonated SAALsSLLR and SAALsYLLR exhibit a
very intense neutral loss of 80 Da, Fig. 1. The sulfo group loss from
the precursor ion as well as from b and y product ions dominates
all tandem MS spectra analysed in this work (Fig. 1 and Fig. S1 –
supplementary data). However, in some cases, small portion of b
and y ions retain the sulfo group and enable characterisation of the
peptides (Fig. 1). Those ions are associated with 5.7% of the total
ion intensity of all identified ions (in this work) and are not readily
observed in tandem MS spectra of sulfonated peptides (e.g. Fig. S1
b and ref. [8–10,16]). Therefore, it is sometimes difficult to obtain
site-specific information on the location of the sulfo group.
Although the tandem MS spectra of peptides described here
represent only an example, together with previous results [9,16]
they indicate that the sulfo group loss is the dominant dissociation
channel in the collisionally activated peptides where ionising pro-
ton can migrate to various sites (the mobile proton environment).
It would be of interest to collect more sulfopeptide tandem MS
spectra obtained on different instruments in order to statistically
characterise fragmentation behaviour but this is out of the scope of
the present paper.
The mechanism, which could be associated with the sulfo group
loss, includes the proton transfer from the sulfo group hydroxyl
to the ester oxygen and consequent formation of the hydroxyl
group and SO3(Scheme 1 a). The mechanism is similar to the one
associated with the loss of metaphosphoric acid in phosphorylated

Fig. 1. CID tandem MS product ion spectra of the doubly protonated (a) SAALsSLLR
and (b) SAALsYLLR. *−80 Da (–SO3).
peptides [7] and was hypothesised for the sulfo group loss from
sulfoserine [18].
The potential energy profile for the sulfo group loss from
SAALsSLLR peptide is shown in Fig. 2, together with the opti-
mised structures of the energy minima and transition state. As
hypothesised, the mechanism includes transfer of the acidic side
Scheme 1. . (a) Proton transfer mechanism and (b) intramolecular nucleophilic
substitution (SN2) mechanism.
Fig. 2. Schematic representation of the potential energy profile of the sulfo group
loss from the doubly protonated SAALsSLLR peptide. The relative energies are calcu-
lated at the B3LYP/TZVP//B3LYP/6-31G(d) level of theory with respect to the most
stable SAALsSLLR conformer found. Magnified structures with interatomic distances
are available as supplementary data (Fig. S7).
chain proton from the sulfo group hydroxyl to the ester oxygen.
In the transition state, the sulfo group is additionally stabilised via
interaction with protonated amino terminus. Although there is a
possibility for activating the ionising proton from the amino termi-
nus during reaction, there is no evidence that the ionising proton is
involved in the mechanism. The protonated amino terminus–sulfo
group interaction could suggest a charge-directed pathway where
the charged amino terminus could make the sulfo hydroxyl group
a more electron deficient and thus a more efficient proton donor.
The role of the ionising proton in the proton transfer mechanism
was further examined with singly protonated TsSQLL peptide (the
mobile proton environment). In TsSQLL peptide, the protonated
amino terminus and the sulfonated serine side chain are very
close; therefore, involvement of ionising proton should be evident.
However, the identical reaction mechanism in protonated TsSQLL
peptide does not require interaction of the sulfo group with the pro-
tonated amino terminus, suggesting the charge remote nature of
the sulfo group loss pathway (Fig. S2 – supplementary data). Thus,
observed interaction in SAALsSLLR peptide can be attributed to the
secondary structure stabilisation via hydrogen bonds. Upon SO3
abstraction from the sulfonated side chain, the side chain residue
takes the form structurally analogous to the unmodified hydroxyl-
containing residue, consistent with the IRMPD observations on
sulfoserine [18]. At this point, SO3molecule probably interacts with
peptide and consequently the ion–molecule complex dissociates
making the neutral loss of SO3a two-step process (abstrac-
tion and ion–molecule complex dissociation), in accordance
with observations of phosphopeptides dissociation dynamics
[26].
Reaction barrier for sulfo group loss of the doubly protonated
SAALsSLLR peptide is 174.8 kJ mol−1. Calculated unimolecular reac-
tion rates (by the RRKM theory) show that reaching ms time range
pathway requires 1180 kJ mol−1while increase up to 1880 kJ mol−1
is needed for ␮s time range (Fig. S3 – supplementary data). Rate
energy dependences of SO3loss pathway match literature available
average internal energy values of similar size peptides (approxi-
mately 900 for ms time range and 1700 kJ mol−1␮s time range)
[27].
3.2. CID of sulfonated peptides – limited proton mobility
The precursor ion and backbone fragments in the low-energy
CID spectra of peptides under the mobile proton environment
exhibit a very intense neutral loss of 80 Da Loss of the sulfo moiety
is even more prominent under the limited proton mobility, Fig. 3.

Fig. 3. CID tandem MS product ion spectra of the singly protonated (a) SAALsSLLR
and (b) SAALsYLLR. *−80 Da (–SO3).
Singly protonated SAALsSLLR and SAALsYLLR peptides preferen-
tially eliminate the modification even before backbone fragmenta-
tion occurs. Deposition of the larger amounts of additional vibronic
energy sometimes may result in additional peptide backbone frag-
mentation, in accordance with already documented findings [16].
Assessment of the potential energy surface of the sulfo group
loss for the singly protonated SAALsSLLR reveals identical charge
remote mechanism as the one under the mobile proton environ-
ment (Fig. 1a). However, there is no interaction between the sulfo
moiety and the arginine guanidino group.
According to RRKM calculations, the sulfo group loss from the
precursor ion occurs at the millisecond time scale at the internal
energy of 1010 kJ mol−1(Fig S4 – supplementary data). Internal
energy window for the peptide bond cleavage is from 1000 to
1700 kJ mol−1[27,28], suggesting elimination of the sulfo moiety
from the precursor ion as the principal dissociation channel, which
is in agreement with our experimental observations.
3.3. Neutral loss of PTM: sulfonated vs. phosphorylated peptides
The major fragmentation pathway occurring from energised
protonated sulfopeptide originates by loss of SO3(−80 Da), Fig. 1. A
phospho modification containing peptides (except pTyr residue),
in contrast, shows pronounced peaks related to elimination of
H3PO4(−98 Da) (tandem MS spectra of TpSQLL and SAALpSLLR
in Fig. S5 – supplementary data). Neutral loss of the phosphoric
acid involves the charge-directed intramolecular nucleophilic sub-
stitution (SN2) mechanism (Scheme 1b) while sulfur trioxide loss
Table 1
The proton transfer and SN2 mechanism reaction barriers (in kJ mol−1) calculated at
the B3LYP/TZVP//B3LYP/6-31G(d) level of theory. The reaction barriers were calcu-
lated with respect to the most stable peptide conformation. Total electronic energies
(in Eh) and atomic coordinates are available in supporting data file (Table S1).
Peptide Charge state Reaction mechanism
Proton transfer SN2
TsSQLL 1 148.9 157.1
TpSQLL 1 165.7 134.6
SAALsSLLR 1 155.1 167.9
SAALpSLLR 1 173.5 166.9
SAALsSLLR 2 174.8 181.2
SAALpSLLR 2 200.4 180.1
Model system (sS)a1 111.1 89.3
Model system (pS)a1 160.6 93
aReaction barrier of the model system was calculated using G3(MP2)//B3LYP
protocol.
is related to charge remote proton transfer mechanism (Fig. 2a). In
both modifications, the presence of other modification dominant
mechanisms can be observed to a smaller extent, i.e. SN2 mecha-
nism in sulfonated peptides (loss of sulfuric acid, −98 Da e.g. Fig.
S1 – supplementary data) and proton transfer mechanism in phos-
phorylated peptides (loss of metaphosphoric acid, −98 Da, e.g. ref.
[6]). Both modifications represent similar highly acidic monoesters
and have potential for proton transfer and intramolecular nucleo-
philic substitution; however during collisional activation different
dissociation channels were dominant.
To compare both mechanisms, potential energy profiles for
phosphorylated/sulfonated SAALSLLR and TSQLL peptides were
constructed and the corresponding reaction barriers at the
B3LYP/TZVP//B3LYP/6-31G(d) level of theory are given in Table 1.
From reactions barriers, it follows that moving from sulfonated
to phosphorylated peptide increases the proton transfer barrier
height while SN2 threshold remains roughly the same or slightly
lower in phosphorylated peptides. An explanation is offered by
inspecting the nature of functional groups.
In the intramolecular nucleophilic substitution reaction, the
alkyl group–ester oxygen bond is cleaved while in the proton trans-
fer reaction, the ester oxygen–sulfur/phosphor bond is cleaved.
SN2 reaction breaks the same bond regardless of PTM while pro-
ton transfer mechanism breaks a different bond. Regarding the
bond strength, it is useful to consider mean bond dissociation
enthalpy since it may provide rough estimate of relative bond
strength. S O bond dissociation enthalpy is 37 kJ mol−1lower
than P O bond dissociation enthalpy, suggesting that S O bond
is weaker than P O[29]. SN2 barrier lowering in peptides bear-
ing phospho modification may be attributed to extra hydroxylic
group available at phospho residue which enables additional
hydrogen bond formation and stabilisation of the transition state
structure. Intramolecular nucleophilic substitution requires side
chain–polypeptide chain interaction while proton transfer mech-
anism is restricted only to the side chain; thus, SN2 mechanism
requires “more defined” transition state conformation and there-
fore it is more susceptible to stabilisation by additional hydrogen
bond. The small model system (see Section 2) can provide indica-
tive information about the barriers taking into account the bond
strength and (on the other hand) it is free of secondary structure
stabilisation effects. SN2 barrier estimated at the sulfo and phospho
model systems is roughly the same while the proton transfer bar-
rier for the phospho model system is ∼50 kJ mol−1which is higher
than for sulfo (Table 1).
In both mechanisms, the geometry of transition state is rel-
atively close to the product geometry and consequently RRKM
curves will have similar shape (Fig. 4 and Fig. S6). Since the size
of the precursor does not change, the main difference between

Fig. 4. RRKM theory unimolecular reaction rate constants for the proton transfer
and SN2 pathway in doubly protonated SAALsSLLR and SAALpSLLR peptide.
RRKM curves will be determined by activation energy (reaction
threshold). Accordingly, change in modification from sulfonation to
phosphorylation will raise proton transfer barrier (and lower SN2
barrier) making nucleophilic substitution mechanism predominant
(Fig. 4 and Fig S6). RRKM plots show dominance of proton transfer
mechanism for sulfonated peptides and dominance of SN2 path-
way in phosphorylated peptides, which is in agreement with our
suggestions and experimental data (Fig. 4 and Fig S6).
4. Conclusions
Combined experimental and theoretical investigation of the gas-
phase dissociation behaviour of sulfonated peptides yielded the
following results.
Regardless of proton mobility environment, neutral loss of 80 Da
from sulfonated peptides is related to the charge remote pro-
ton transfer mechanism. Molecular modelling predicts that during
vibrational excitation, the acidic side chain proton is transferred
from sulfo group hydroxyl to the ester oxygen. Protonation of
the ester oxygen leads to S O bond cleavage and to formation of
unmodified hydroxyl containing residue and SO3.
In comparison with other dissociation channels (e.g. b/y ion for-
mation), the neutral loss product ion(s) should be dominant in the
tandem MS spectrum. Furthermore, with limiting proton mobility,
their abundance should increase even more.
Although identical dissociation pathways (the proton transfer
and the intramolecular nucleophilic substitution) are available for
sulfonated and phosphorylated peptides, each PTM activates dif-
ferent mechanism. Preference of the proton transfer mechanism
in sulfonated peptides is a consequence of the fact that the S O
bond is weaker compared to P O bond. On the other hand, due to
the stronger P O bond and the possibility of additional stabilisa-
tion of the transition state (additional OH group), phosphorylated
peptides follow the intramolecular nucleophilic substitution
mechanism.
Acknowledgements
This manuscript is dedicated to Prof. Simon J. Gaskell in happy
celebration of his 65th birthday. The Ministry of Science, Education
and Sports of Republic of Croatia supported this work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijms.2015.07.023
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