Fragmentation behavior of a thiourea-based reagent for protein structure analysis by collision-induced dissociative chemical cross-linking

Article abstract of DOI:10.1002/jms.1775

The fragmentation behavior of a novel thiourea-based cross-linker molecule specifically designed for collision-induced dissociation (CID) MS/MS experiments is described. The development of this cross-linker is part of our ongoing efforts to synthesize novel reagents, which create either characteristic fragment ions or indicative constant neutral losses (CNLs) during tandem mass spectrometry allowing a selective and sensitive analysis of cross-linked products. The new derivatizing reagent for chemical cross-linking solely contains a thiourea moiety that is flanked by two amine-reactive N-hydroxy succinimide (NHS) ester moieties for reaction with lysines or free N-termini in proteins. The new reagent offers simple synthetic access and easy structural variation of either length or functionalities at both ends. The thiourea moiety exhibits specifically tailored CID fragmentation capabilities - a characteristic CNL of 85 u - ensuring a reliable detection of derivatized peptides by both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry and as such possesses a versatile applicability for chemical cross-linking studies. A detailed examination of the CID behavior of the presented thiourea-based reagent reveals that slight structural variations of the reagent will be necessary to ensure its comprehensive and efficient application for chemical cross-linking of proteins. Copyright

Full text of DOI:10.1002/jms.1775

Research Article
Received: 12 April 2010
Accepted: 27 May 2010
Published online in Wiley Interscience: 7 July 2010
(
www.interscience.com) DOI 10.1002/jms.1775
Fragmentation behavior of a thiourea-based
reagent for protein structure analysis by
collision-induced dissociative chemical
cross-linking
a
b
a
b∗
Mathias Q. M u¨ ller, Frank Dreiocker, Christian H. Ihling, Mathias Sch a¨ fer
a∗
and Andrea Sinz
The fragmentation behavior of a novel thiourea-based cross-linker molecule specifically designed for collision-induced
dissociation (CID) MS/MS experiments is described. The development of this cross-linker is part of our ongoing efforts to
synthesize novel reagents, which create either characteristic fragment ions or indicative constant neutral losses (CNLs) during
tandem mass spectrometry allowing a selective and sensitive analysis of cross-linked products. The new derivatizing reagent
for chemical cross-linking solely contains a thiourea moiety that is flanked by two amine-reactive N-hydroxy succinimide
(
NHS) ester moieties for reaction with lysines or free N-termini in proteins. The new reagent offers simple synthetic access
and easy structural variation of either length or functionalities at both ends. The thiourea moiety exhibits specifically tailored
CID fragmentation capabilities – a characteristic CNL of 85 u – ensuring a reliable detection of derivatized peptides by both
electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry and as such
possessesaversatileapplicabilityforchemicalcross-linkingstudies.AdetailedexaminationoftheCIDbehaviorofthepresented
thiourea-based reagent reveals that slight structural variations of the reagent will be necessary to ensure its comprehensive
and efficient application for chemical cross-linking of proteins. Copyright ꢀc 2010 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: chemical cross-linking; protein structure analysis; CID; CNL
Introduction
MS/MS. Yet, the synthesis of this ‘first-generation’ cleavable cross-
linker proved to be quite challenging. Therefore, we extended
the thiourea-based concept of a dissociative cross-linker toward
a simplified symmetrical thiourea analog, which is presented
herein. The characteristic fragmentation of the thiourea-based
reagent is directed by the influence of the thiourea moiety
that is incorporated into the reagent. The applicability of our
novel reagent as chemical cross-linker was examined with a set
of model compounds, i.e. substance P (amino acid sequence
RPKPQQFFGLM-NH2), luteinizing hormone-releasing hormone
Chemicalcross-linkingcombinedwithmassspectrometrypresents
an emerging approach to study three-dimensional protein
structuresandtomapproteininterfaces.
[
1–4]
Yet,anunambiguous,
sensitive, reliable and rapid identification of the amino acids
involved in the covalent derivatization by chemical cross-linking
still poses a challenge to the scientist. A mass spectrometric
identification of the chemically modified amino acids in the
respective protein is performed after enzymatic digestion of
the cross-linking reaction mixture. This is often hampered by
the complexity of the created peptide mixtures as often only a
relatively small percentage of cross-linked products are present
besides a large majority of unmodified peptides. One strategy
for a facilitated mass spectrometric analysis of cross-linked
products relies on the application of cross-linkers that fragment
readily during collision-induced dissociation (CID) and give rise to
(LHRH, amino acid sequence pEHWSYGLRPG) as well as the
proteins hen egg lysozyme (14.3 kDa) and the ligand binding
∗
Correspondence to: Andrea Sinz, Department of Pharmaceutical Chemistry
and Bioanalytics, Institute of Pharmacy, Martin-Luther-Universit a¨ t Halle-
Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany.
E-mail: andrea.sinz@pharmazie.uni-halle.de
[
5–8]
characteristic fragment ions.
[
9]
In a previous publication, we reported the synthesis and
application of a novel dissociative chemical cross-linker with a
thiourea moiety, containing a highly nucleophilic sulfur for an
attack at the adjacent glycylproline amide carbonyl. This structural
feature allows for efficiently initiating cleavage of the cross-linker
molecule upon CID. The characteristic fragmentation of the cross-
linker molecule delivered highly indicative mass shifted product
ions and constant neutral losses (CNLs), enabling a sensitive,
selective and unambiguous detection of cross-linked species by
Mathias Sch a¨ fer, Institute of Organic Chemistry, Department of Chemistry,
Universit a¨ t zu K o¨ ln, Greinstr. 4, D-50939 K o¨ ln, Germany.
E-mail: mathias.schaefer@uni-koeln.de
a Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Phar-
macy, Martin-Luther-Universit a¨ t Halle-Wittenberg, Wolfgang-Langenbeck-Str.
4, D-06120 Halle (Saale), Germany
b Institute of Organic Chemistry, Department of Chemistry, Universit a¨ t zu K o¨ ln,
Greinstr. 4, D-50939 K o¨ ln, Germany
J. Mass. Spectrom. 2010, 45, 880–891
Copyright ꢀc 2010 John Wiley & Sons, Ltd.

Fragmentation behavior of a thiourea-based cross-linker
domain of the peroxisome proliferator-activated receptor alpha
layers were dried over magnesium sulfate and the solvent was
removed under reduced pressure to give 4-[3-(3-carboxy-propyl)-
thioureido]-butyric acid (5) as a colorless solid at a yield of 92%.
[
10]
(
PPARα, ∼32 kDa) with ESI (electrospray ionization) and matrix-
[11]
[17]
assisted laser desorption/ionization (MALDI)
tandem mass
spectrometry. The fragmentation behavior of the CID cleavable
cross-linker was examined over a wide range of activation energies
using both a linear quadrupole ion trap and a TOF/TOF instrument.
For activation of the two carboxylic acid functionalities to give
the N-hydroxy succinimide (NHS) ester, 5 was dissolved in pyridine
and treated with N-trifluoroacetoxy succinimide (6) – prepared
from NHS and trifluoroacetic anhydride[ – at 20 C. After 1 h,
the solution was poured into diethyl ether and the precipitate
was removed by filtration. The solid was washed with diethyl
ether and dissolved in a mixture of dichloromethane and toluene.
After the removal of the solvent under reduced pressure, the
residue was dried under high vacuum to give 4-{3-[3-(2,5-dioxo-
pyrrolidin-1-yloxycarbonyl)-propyl]-thioureido}-butyric acid 2,5-
18]
◦
Experimental
Materials
All chemicals and solvents were used as purchased without further
purification (Acros Organics, Geel, Belgium; ABCR, Karlsruhe,
Germany).Tolueneanddiethyletherweredriedbydistillationfrom
benzophenone and sodium, dichloromethane and pyridine by
distillation from calcium hydride. All other solvents were distilled
over a column prior to use. Hen egg lysozyme, substance P, LHRH
and buffer reagents were obtained from Sigma (Taufkirchen,
Germany). The protease bovine trypsin was obtained from Roche
Diagnostics (Mannheim, Germany). Nano-HPLC solvents were of
spectrometric grade (Uvasol, VWR, Darmstadt, Germany). MALDI
matrices and calibration standards were purchased from Bruker
Daltonik (Bremen, Germany). Water was purified with a Direct-
Q5 water purification system (Millipore, Eschborn, Germany). The
ligand binding domain of PPARα was expressed in Escherichia coli
and purified according to a previously published protocol.[
[
19]
dioxo-pyrrolidin-1-yl ester (7) as a colorless oil at a yield of 93%.
Cross-linking reactions
For cross-linking experiments, aqueous stock solutions of sub-
stance P (100 µg/ml), LHRH (100 µg/ml), lysozyme (10 mg/ml)
or PPARα ligand binding domain (2 mg/ml) were diluted
with 20 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid
(
HEPES) buffer (pH 7.4) to give 1 ml solution containing the pro-
tein/peptide at a concentration of 10 µM. The cross-linker (200 mM
stock solution in dimethylsulfoxide) was added in 50-, 100- and
2
00-fold molar excess to the protein/peptide solution and the
12]
reactions were allowed to proceed for 5, 15, 30, 60 and 120 min.
Reactions were quenched with ammonium bicarbonate (20 mM
final concentration). One 200-µl aliquot was taken from each
Synthesis of the thiourea cross-linker
◦
sample and stored at −20 C before MS analysis. Cross-linked
Esterification of 4-amino-butyric acid (1, Scheme S1, Supporting
Information) was achieved by reaction with thionyl chloride in
methanol for 16 h. After the evaporation of the solvent from the
reaction mixture, the crude product was stirred in ethyl acetate.
The hydrochloride of 4-amino-butyric acid methyl ester (2) was
isolated by filtration as a colorless solid at a yield of 98% (Scheme
S1).[
lysozyme and PPARα were digested with trypsin (1 : 100 (w/w)
◦
enzyme to substrate ratio) overnight at 37 C according to an
existing protocol.[ The resulting digests were stored at −20 C
20]
◦
before MS analysis.
MALDI-TOF mass spectrometry linear mode
13]
For the synthesis of 4-isothiocyanato-butyric acid methyl ester
3), 2 was suspended in a mixture of dichloromethane and carbon
MALDI-TOF mass spectrometry of intact cross-linked lysozyme
and PPARα was performed in linear and positive ionization mode
on an Ultraflex III instrument (Bruker Daltonik, Bremen, Germany)
equipped with a Smart beam laser using sinapinic acid as matrix.
Five picomole of each sample was applied onto a ground steel
target using the double layer method.
(
disulfide. After slowly adding triethylamine, the reaction mixture
was stirred for 30 min before methyl chloroformate was added.

◦
After refluxing for 4 h, the mixture was cooled down to 20 C and
poured into water. The organic layer was washed with diluted
hydrochloric acid and brine, dried over magnesium sulfate and
concentrated under reduced pressure. The resulting yellowish oil
Nano-HPLC/MALDI-TOF/TOF mass spectrometry
was distilled in high vacuum to give 3 as a colorless oil at a yield of
14]
8
0%.[
Tryptic peptide mixtures of lysozyme and PPARα were analyzed
by offline coupling of a nano-HPLC system (Ultimate 3000,
Dionex, Idstein, Germany)toaMALDI-TOF/TOFmassspectrometer
(Ultraflex III), whereas the peptides substance P and LHRH were
analyzed by MALDI-TOF/TOF-MS/MS without a preceding nano-
HPLC separation. Samples were injected onto a precolumn
(Acclaim PepMap, C18, 300 µm × 5 mm, 5 µm, 100 Å, Dionex)
and desalted by washing the precolumn for 15 min with 0.1%
trifluoroacetic acid (TFA) before the peptides were eluted onto the
separationcolumn(AcclaimPepMap, C18, 75 µm×150 mm, 3 µm,
100 Å,Dionex),whichhadbeenequilibratedwith95%solventA(A:
5% acetonitrile (ACN), 0.05% TFA). Peptides were separated with
a 60-min gradient (0–60 min: 5–50% B, 60–62 min: 50–100% B,
62–67 min: 100% B, 67–72 min: 5% B, with solvent B: 80% ACN,
0.04% TFA) at a flow rate of 300 nl/min with UV detection at 214
and 280 nm. Eluates were fractionated into 18-s fractions using the
fraction collector Proteineer fc (Bruker Daltonik), mixed with 1.1 µl
For the reaction of the isothiocyanate 3 and the protected
amine 2, the amine was suspended in dichloromethane and
◦
treated with triethylamine. After 15 min, 3 was added at 0 C
◦
and the reaction mixture was brought to 20 C within 3 h. After
washingwithdilutedhydrochloricacidandbrine, theorganiclayer
was dried over magnesium sulfate and the solvent was removed
under reduced pressure. The resulting yellowish oil was purified
by column chromatography with ethyl acetate/cyclohexane [1 : 2
(
v/v)] to give 4-[3-(3-methoxycarbonyl-propyl)-thioureido]-butyric
[
15,16]
acid methyl ester (4) as colorless oil at a yield of 91%.
Hydrolysis of the two methyl ester functionalities of 4 was
achieved with lithium hydroxide in a mixture of tetrahydrofurane,
◦
methanol and water for 15 h at 20 C. After the removal of
the solvent under reduced pressure, the residue was dissolved
in water and acidified to pH 2 using 2 M hydrochloric acid.
After seven extractions with ethyl acetate, the combined organic
J. Mass. Spectrom. 2010, 45, 880–891
Copyright ꢀc 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

M. Q. M u¨ ller et al.
of matrix solution (0.8 mg/ml α-cyano-4-hydroxy cinnamic acid Results and Discussion
in 90% ACN, 0.1% TFA, 1 mM NH4H2PO4) and directly prepared
onto a 384 MTP 800 µm AnchorChip target (Bruker Daltonik).
MALDI-TOF/TOF-MS analyses were conducted in the positive
ionization and reflectron mode by accumulating 2000 laser shots
in the range m/z 800–5000 to one mass spectrum. Mass spectra
were externally calibrated using Peptide Calibration Standard II
Cross-linker design
Our novel dissociative thiourea cross-linker was developed on
the basis of a previously synthesized cross-linker containing
a thiourea moiety that is connected to proline via a glycine
[9]
residue. This previous analytical concept relied on the lability
(
Bruker Daltonik). Signals with a signal-to-noise ratio (S/N) >15
were isolated for laser-induced fragmentation. In-source decay
ISD) was performed manually on isolated cross-linked product
of the glycylproline bond, which is preferably cleaved by an
intramolecular attack of the strongly nucleophilic thiocarbonyl
sulfur.Thisattackispromotedbythepreferablyprotonatedproline
amide nitrogen. Although we proved the general feasibility of the
proposed concept, the synthesis of that ‘first-generation’ collision-
induced dissociative cross-linker proved to be quite challenging,
which motivated us to develop a more easily accessible thiourea-
containingcross-linker.Suchanovelandsimplifiedthiourea-based
reagent should exhibit specifically tailored CID fragmentation
capabilities, thus ensuring a sensitive and selective detection of
derivatized peptides. Moreover, we aimed to examine whether
the presence of a proline is essential for the preferred cleavage
of the cross-linker. Hence, the novel compound solely contains a
thiourea moiety flanked by two amine-reactive NHS ester moieties
(
candidates by increasing the laser energy by ca 10% relative to the
usual laser energy in LID (laser-induced dissociation) MS/MS. Data
acquisition was done automatically by the WarpLC 1.1 software
(Bruker Daltonik) coordinating MS data acquisition (FlexControl
1.3) and data processing (FlexAnalysis 3.0) softwares.
Nano-HPLC/Nano-ESI-LTQ-Orbitrap mass spectrometry
Fractionation of tryptic peptide mixtures (lysozyme and PPARα)
was carried out on an Ultimate nano-HPLC system (Dionex
Corporation, Idstein, Germany) using reversed phase C18 columns
(
precolumn: Acclaim PepMap, 300 µm × 5 mm, 5 µm, 100 Å,
separation column: Acclaim PepMap, 75 µm × 150 mm, 3 µm,
00 Å, Dionex Corp., Idstein, Germany). After washing the
(
Scheme 1), which react with lysines or free N-termini in proteins.
The reactivity of our novel cross-linker is comparable to classical
NHS ester-based reagents, such as bis(sulfosuccinimidyl) suberate.
The abbreviation BuTuBu for the novel designed linker that is
used throughout this article is deduced from thiourea (Tu) and
amino butyric acid (Bu) (Scheme 1). The reaction scheme of the
thiourea cross-linker is presented in Scheme 2. After one reactive
site has reacted with a primary amine in a protein, the second
reactive site might react with another amine group forming
an intramolecular cross-link (1). Alternatively, the second NHS
functionality either stays intact or is hydrolyzed or aminolyzed
during reaction quenching (2). For quenching the cross-linking
reaction, ammonium salts, i.e. NH4HCO3, are added. From initial
experiments,itimmediatelyturnedoutthatintramolecularlycross-
1
precolumn for 15 min with water containing 0.1% TFA, the
peptides were eluted and separated using a gradient from 0
to 50% B (90 min), 50 to 100% B (1 min) and 100% B (5 min),
with solvent A: 5% ACN containing 0.1% formic acid (FA) and
solvent B: 80% ACN containing 0.1% FA. The nano-HPLC system
was directly coupled to the nano-ESI source (Proxeon, Odense,
Denmark) of an LTQ (linear ion trap)-Orbitrap XL hybrid mass
spectrometer (ThermoFisher Scientific, Bremen, Germany). MS
2
data were acquired over 122 min in a data-dependent MS mode:
eachhigh-resolutionfullscan(m/z 300–2000, resolutionwassetto
6
0 000) in the orbitrap was followed by three product ion scans in
the orbitrap (resolution 15 000) for the three most intense signals
+
linkedspecies[M+H+212 u] (1)areaccompaniedbyanisobaric
3
in the full-scan mass spectrum (isolation window 1.5 u). MS in the
cyclic 2-alkylylimino-[1,3]thiazepan-7-one (3). The latter is formed
upon collision activation by rearrangement of (1), but might also
originate from a ‘dead-end’ cross-linked species (2), i.e. a peptide
thathasbeenmodifiedbyapartiallyhydrolyzedcross-linker(type0
2
orbitrap was performed on the two mostintense signals in the MS
spectra with a resolution of 15 000. Dynamic exclusion (exclusion
duration 180 s, exclusion window −1 to 2 Th (Thomson [m/z])) was
enabled to allow detection of less abundant ions. Data acquisition
was controlled via XCalibur 2.0.7 (ThermoFisher) in combination
with DCMS link 2.0 (Dionex).
[24]
cross-link). Eventually, the rearrangement of (3) leads to the loss
of a γ -lactame (85 u) to form the 7-alkylylimino-[1,3]oxazepane-2-
thione derivative (4). This reaction is perfectly suited for a general
identification of BuTuBu-derivatized species and greatly facilitates
the identification of cross-linked peptides.
Offline nano-ESI-LTQ-Orbitrap-MS
Cross-linked substance P and LHRH were additionally analyzed
by offline nano-ESI-LTQ-Orbitrap-MS, with the resolution in the
3
orbitrap set to 100 000. MS/MS and MS experiments were
conducted with a relative collision energy in the linear ion trap
(
LTQ) of 35% and selected product ions were analyzed in the
orbitrap analyzer set to a resolution of 100 000.
Analysis of cross-linked products
Cross-linked products were identified by analyzing the MS
data using General Protein Mass Analysis for Windows
GPMAW)[ version 8.10 (Lighthouse Data, Odense, Denmark,
21]
(
http://www.gpmaw.com) and the CoolToolBox software program,
which is an upgrade of the VIRTUALMSLAB software program.[
The length of the cross-linker was calculated using the Viewer
Scheme 1. Structure of the novel homobifunctional chemical cross-linker
NHS–BuTuBu–NHS. The abbreviations are indicated using parenthesis.
22]
[
23]
Light 5.0 software (Accelrys).
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Copyright ꢀc 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 880–891

Fragmentation behavior of a thiourea-based cross-linker
Scheme 2. Reactions of the amine-reactive thiourea cross-linker NHS–BuTuBu–NHS.
Cross-linking of substance P
(structure 4 in Scheme 2). To explain the characteristic and
abundant loss of 85 u, we propose a rearrangement reaction
For initial experiments with the thiourea cross-linker, the 11-
amino acid peptide substance P was employed as it contains
merely two reactive sites for the amine-reactive thiourea cross-
linker, the lysine residue at position three of the peptide and the
free N-terminus. Therefore, substance P presents a straightforward
model system to study the properties of the novel cross-linker.
The fragmentation behavior was evaluated in both ESI and
MALDI mass spectrometric experiments. In Fig. 1A, the MALDI-
TOF mass spectrum of substance P after reaction with the
thiourea cross-linker is presented. From the MALDI spectrum
alone it is not clear whether the cross-linker molecule is attached
forming both the neutral γ -lactame (85 u) and a [M + BuTu
+
+
H] (i.e. 7-alkylimino-[1,3]oxazepane-2-thione derivative (4) in
Scheme 2). Yet, from MALDI-MS/MS data alone it was not possible
to definitely assign the modification to Lys-3, the N-terminus or
an intramolecular cross-link between both amine groups, nor is
it possible to rule out the possibility of having mixed isomeric
species of the named options. This is due to the absence of a
characteristic b2 product ion, while the observed product ions
are indicated in the insets of Fig. 1B. However, the dominant
abundance of the product ion corresponding to the CNL of 85 u
(Fig. 1B) clearly demonstrates that the respective intramolecular
to Lys-3 or to the N-terminus. Strikingly, the signals at m/z
+
nucleophilic attack of the thiocarbonyl sulfur definitely presents
the preferred fragmentation channel, confirming the validity of
the basic concept of our novel cross-linker. Further information
on the created product ion is obtained by additionally conducted
MALDI-ISD (in-source decay) MS/MS experiments (Fig. 1C).
1
559.834 for the protonated species [M + H + 212 u] and
+
at m/z 1581.827 for the sodium adduct [M + Na + 212 u]
were found to correspond to cross-linker-modified substance
P. At this point, it is important to note that the identity of
the derivatized peptide molecular ions observed in Fig. 1A is
somewhat ambiguous (see Scheme 2): either an intramolecular
cross-link between the N-terminus and the side chain amine group
of lysine is formed (structure 1 in Scheme 2) or alternatively, the
cross-linker has underwent an intramolecular cyclization to form
a seven-membered 2-alkylylimino-[1,3]thiazepan-7-one (structure
In order to evaluate the potential of the thiourea cross-linker for
ESI mass spectrometry, LTQ-Orbitrap mass spectrometric analyses
(
MS and MS/MS) were conducted (Fig. 2). In contrast to the MALDI
mass spectrum, the signal of the doubly charged peptide that is
modified with a cross-linker is visible at m/z 780.407 in the ESI mass
spectrum(Fig. 2A).Inaddition,thesignalsofthetriplychargedions
3
in Scheme 2). This characteristic cyclization reaction depends
3
+
on the pronounced nucleophilicity of the thiocarbonyl sulfur,
which intramolecularly attacks the adjacent carbonyl, leading
to the formation of the seven-membered heterocycle (structure
[M + BuTuBu + 2H + K] at m/z 533.256 and [M + BuTuBu + 2H +
3
+
Na] at m/z 527.930 are detected. Yet, the signal with the highest
abundance is the species at m/z 780.407 with a mass increase
of 212 u compared to the unmodified peptide, indicating the
derivatization with the cross-linker. In order to definitely elucidate
the identity of this ion, CID experiments were performed in the
linear ion trap (LTQ), whereas exact ion mass analysis of fragment
ions was performed with high resolution (RFWHM = 15 000) in
3
in Scheme 2). According to Scheme 2, a mass increase of
2
12 u compared to the peptide is observed for the cross-linked
product. The signal at m/z 1559.834 was isolated and examined
by CID in the MALDI-TOF/TOF mass spectrometer resulting in
a product ion at m/z 1474.7 that arises from a CNL of 85 u
J. Mass. Spectrom. 2010, 45, 880–891
Copyright ꢀc 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

M. Q. M u¨ ller et al.
+
Figure 1. (A) MALDI-TOF mass spectrum of substance P ([M + H] at m/z 1347.746) after cross-linking reaction with the thiourea linker, resulting in a
mass shift of 212 u (reaction according to Scheme 2). Please note that a number of signals originating from impurities in the substance P sample are
+
visible in the mass spectrum. The ion at m/z 1575.883 is labeled as [Mox + BuTuBu + H] referring to oxidized substance P after cross-linking reaction
with the thiourea linker (mass shift of 16 u). Asterisks indicate impurities in substance P. (B) CID fragment ion mass spectrum (MALDI-TOF/TOF-MS/MS) of
the signal at m/z 1559.834. The respective isolated and fragmented species are presented in the insets. (C) ISD-MALDI fragment ion mass spectrum of [M
+
BuTu + H]+ at m/z 1474.8. $: signal is not assigned.
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Copyright ꢀc 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 880–891

Fragmentation behavior of a thiourea-based cross-linker
Figure 2. (A) ESI-LTQ-Orbitrap mass spectrum of substance P after cross-linking reaction with the thiourea cross-linker. (B) CID fragment ion mass
spectrum (MS/MS) of the signal at m/z 780.407 showing substance P modified with BuTuBu (Scheme 2, structure 1 or 3). (C) CID fragment ion mass
3
spectrum (MS ) of the signal at m/z 706.37. The signals assigned with $ are frequently observed in the spectra obtained with our LTQ-Orbitrap mass
spectrometer.
J. Mass. Spectrom. 2010, 45, 880–891
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M. Q. M u¨ ller et al.
Figure 3. Linear MALDI-TOF mass spectrum of lysozyme with different excess of cross-linker and reaction times. The average molecular weight of
protonated hen egg lysozyme (14 313 Da) matches perfectly with the observed signal at m/z 14 313.
the orbitrap. The ion at m/z 780.407 (either structure 1 or 3)
loses 85 u to form the product ion (4) at m/z 737.88. Furthermore,
sequenceanalysisonthebasisoftheESI-LTQ-OrbitrapCIDtandem
mass spectrum allowed an unambiguous assignment of the cross-
linker modification to the N-terminus of substance P (Fig. 2B).
In particular, the presence of y9- and y10-NH3 ions indicate an
unmodified lysine at position 3 and a cyclized seven-membered
of a modified species with the characteristic mass shift of 212 u
at m/z 1395.682. MS/MS data revealed the indicative CNL of 85 u
both in MALDI-TOF/TOF and ESI-LTQ-Orbitrap mass spectrometry
(data not shown).
Cross-linking of lysozyme
7
-alkylylimino-[1,3]oxazepane-2-thione cross-linker (4) (inset of
For evaluating the applicability of the thiourea cross-linker for
proteins, the 14.3 kDa protein lysozyme was derivatized with our
novelcross-linker.Efficientcross-linkingwasprovenbyMALDI-TOF
massspectrometricmeasurementsinthelinearmodeshowingthe
attachment of up to six – possibly seven – cross-linker molecules
to the protein depending on molar excess (50-, 100- and 200-
fold excess) of cross-linker and reaction times ranging between
Fig. 2B). However, the absence of any substance P ions showing
[
24]
two ‘dead-end’ (type 0) linker molecules attached to both the
N-terminus and the Lys at position 3 could give a hint for the
presence of an intramolecular cross-link of the respective amine
functionalities. Itcanbehypothesizedthatduringthecross-linking
reaction, an intramolecular (type 1) cross-link has been created,
butthatduringCID,aconversionoftype1intoatype0cross-linked
species occurred. We are neither able to exclude nor to verify any
of the potential cross-linker-modified species that are created by
the thiourea cross-linker. This certainly presents a drawback for a
comprehensiveapplicationofthisreagentforcross-linking studies
of proteins.
5
and 60 min (Fig. 3). For gaining insight into the reaction sites
of the cross-linker at the protein, modified lysozyme was in-
solution digested with trypsin and the resulting peptide mixtures
wereanalyzedbynano-HPLC/MALDI-TOF/TOF-MS(/MS)andnano-
HPLC/nano-ESI-LTQ-Orbitrap-MS(/MS).
Exemplarily, a lysozyme peptide comprising amino acids
1
15–125 (CKGTDVQAWIR) was thoroughly analyzed as it contains
a reaction site at lysine in position 116 and another – yet less
likely – reaction site at the threonine residue in position 118. From
theMALDI-TOFmassspectrumpresentedinFig. 4A, amodification
with the thiourea linker is apparent as a molecular ion of the
peptide 115–125 with the characteristic mass shift of 212 u is
observed at m/z 1545.740. This species is accompanied by an
ISD fragment at m/z 1460.661, corresponding to the characteristic
neutral loss of a γ -lactame with 85 u and the formation of the
[1,3]oxazepane-2-thione derivative (see structure 4 in Scheme 2).
The MALDI mass spectrum additionally exhibits two abundant
Cross-linking of LHRH
For monitoring the side reactivity with amino acids containing
hydroxyl groups, cross-linking experiments were performed with
LHRH as it contains two potentially reactive amino acids Ser-4
and Tyr-5. It has been shown that NHS esters also react with
hydroxyl groups in serines, threonines and tyrosines at pH 7.4,
albeit with a lower frequency compared to lysines.
the pyroglutamate the N-terminus cannot be modified. The base
peak showed unmodified LHRH at m/z 1183.576 and a lower yield
[
25,26]
Due to
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J. Mass. Spectrom. 2010, 45, 880–891

Fragmentation behavior of a thiourea-based cross-linker
+
Figure 4. (A) MALDI-TOF mass spectrum of a nano-HPLC fraction containing a lysozyme peptide (amino acids 115–125, [M + BuTuBu + H] at m/z
1
545.740) that is modified with the cross-linker. Please note that Cys-115 is carbamidomethylated. A signal at m/z 1460.661 corresponds to a species
that is created after a CNL of 85 u from the species at m/z 1545.740. A signal at m/z 1471.762 could not be assigned so far. Also visible in the spectrum
is a lysozyme peptide (amino acids 97–114) at m/z 2073.947. (B) CID fragment ion mass spectrum (MALDI-TOF/TOF-MS/MS) of the cross-linker-modified
+
peptide (m/z 1545.740). (C) ISD-MALDI fragment ion mass spectrum of [M + BuTu + H] at m/z 1460.661. (D) Nano-ESI-LTQ-Orbitrap mass spectrum of a
2+
nano-HPLC fraction containing a cross-linker-modified lysozyme peptide (amino acids 115–125, [M + BuTuBu + 2H] at m/z 773.369). (E) CID fragment
ion mass spectrum (nano-ESI-LTQ-Orbitrap-MS/MS) of the signal at m/z 773.369, fragmentation in the LTQ, analysis of fragment ions in the orbitrap. The
3
isolated and fragmented species are represented in the insets. (F) CID fragment ion mass spectrum (MS ) of the signal at m/z 764.36. The isolated and
fragmented species are represented in the insets; $: signal is not assigned.
ions at m/z 2073.947 and 1471.762. The signal at m/z 2073.947
was assigned as lysozyme peptide composed of amino acids
Inthefollowing, differenttypesofcross-linkedproducts, namely
modified molecular ions mass shifted by 212 u (either intramolec-
ularly cross-linked (type 1) peptides (structure 1 in Scheme 2)
or peptides modified by the intramolecularly cyclized seven-
membered 2-alkylylimino-[1,3]thiazepan-7-one (type 0 cross-link);
9
7–114 based on MS/MS experiments. The ion at m/z 1471.762
unambiguously results from an ISD fragmentation of the cross-
linked precursor ion at m/z 1545.740 as the amino acid sequence
of the ion at m/z 1471.762 was elucidated to be identical to that
of the species at m/z 1545.7 and confirmed by additional MS/MS
experiments (data not shown). The fragmentation mechanism and
the mass difference of 74 u, formally corresponding to the loss of
SOCN, are still a matter of debate.
[
24]
structure 3 in Scheme 2) and type 2 (interpeptide) cross-links
were analyzed both in MALDI and ESI-CID MS/MS experiments for
gaining insight into the specific fragmentation patterns for each
type of cross-linked product. In Fig. 4B, the product ion spectrum
(MALDI-TOF/TOF-MS/MS) of the molecular ion with the character-
J. Mass. Spectrom. 2010, 45, 880–891
Copyright ꢀc 2010 John Wiley & Sons, Ltd.
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M. Q. M u¨ ller et al.
Figure 4. (continued).
istic mass shift of 212 u at m/z 1545.740 is presented (structure 3).
Due to the presence of the C-terminal arginine, exclusively y-type
ionsarevisibleinthefragmentionmassspectrum,thusunambigu-
ouslydemonstratingthemodificationatlysine-116.Byfarthemost
prominent signal in the MALDI-CID tandem mass spectrum is the
signal at m/z 1460.6, i.e. the 7-alkylimino-[1,3]oxazepane-2-thione
Finally, an interpeptide (type 2) cross-linked product[24] within
lysozyme connecting the N-terminal lysine with lysine-97 was
investigated. According to the crystal structure of hen egg
lysozyme (pdb entry 193L) both N-atoms are at a distance of
23.2 Å and can readily be cross-linked by the thiourea linker that
possesses a length of 12.5 Å due to the inherent flexibility of the
lysinesidechains.Inpreviouslyconductedcross-linkingstudies,we
foundbothlysinestobepotentialreactionsitesofNHSester-based
cross-linkers.[ The MALDI-TOF mass spectrum shows the cross-
linked product at m/z 2144.023 (Fig. 5A). MALDI-TOF/TOF-MS/MS
analyses of this species revealed several y-type ions (Fig. 5B),
whereas ESI-LTQ-Orbitrap-MS/MS also yielded a number of b-type
ions (Fig. 5C). The interpeptide cross-link was thus unambiguously
identified based on a number of fragment ions of the longer
lysozyme peptide constituting the cross-linked product. The fact
that the connected lysine represents only a single amino acid is
readily explained as it represents the N-terminal amino acid of
lysozyme. All other lysines in the amino acid sequence of lysozyme
would not have been created as single amino acid after tryptic
cleavage. In order for trypsin to cleave C-terminal to Lys-1, the
(4) that had been created by a CNL of 85 u from the precursor
ion (3) (Fig. 4B). Additionally conducted MALDI-ISD MS/MS exper-
iments of the latter species yielded a complete series of y-type
ions and an almost complete b-type ion series (Fig. 4C). From the
mass difference between the y9 and y10 ions, the modification
was unambiguously assigned to the lysine in position 116. As for
27]
3
substance P, ESI-LTQ-Orbitrap-MS, MS/MS and MS data were ob-
tained for tryptic peptides derived from the digestion of lysozyme.
The respective spectra are shown in Fig. 4D–F. The peptide com-
prising amino acids 115–125 that is modified with a cross-linker
molecule is visible as doubly charged signal at m/z 773.369 in the
3
nano-ESI-LTQ-Orbitrap mass spectrum (Fig. 4D). MS/MS and MS
data confirm the modification with the cross-linker at lysine-116
(Fig. 4E and F).
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J. Mass. Spectrom. 2010, 45, 880–891

Fragmentation behavior of a thiourea-based cross-linker
Figure 4. (continued).
cross-linking reaction must have occurred at the N-terminal amine
group instead of the ε-amine group of the lysine side chain – a
behavior that we frequently observe when cross-linking lysozyme.
applicability of the thiourea compound as dissociative linker relies
solely on the nucleophilicity of the thiocarbonyl sulfur, which is
responsible for an efficient and preferred dissociation upon CID.
The fragmentation behavior of the novel compound was probed
under low-energy CID conditions (kinetic energy of the precursor
ion <100 eV) in a linear quadrupole ion trap as well as under
high-energy CID conditions (kinetic energy of the precursor ion in
the range of keV) in a TOF/TOF system with respectively modified
peptide ions. The results clearly confirm the feasibility of the
proposed analytical concept as the amide bond adjacent to the
thiocarbonyl sulfur is more easily cleaved than any other peptide
backbone bond leading to the loss of a γ -lactame (85 u) and the
formation of a 7-alkylimino-[1,3]oxazepane-2-thione (structure 4
in Scheme 2). On the basis of this characteristic 85 u-CNL, a
rapid identification of peptide ions that are modified with the
derivatizing agent is realized. However, our present investigations
reveal that the fragmentation behavior of the thiourea compound
is not unambiguous (Scheme 2), which hampers a general
application of this particular reagent as a chemical cross-linker
for protein structural studies. Current investigations with a
number of thiourea compounds are underway to circumvent
the apparent challenges that are inherently associated with the
reagent described herein.
Cross-linking of PPARα
As for lysozyme, MALDI- and ESI-MS and CID-MS/MS experiments
were conducted for the 32-kDa ligand binding domain PPARα.[
From the b- and y-type ions of a PPARα peptide (amino acids
11]
2
13–222) in CID-MS/MS, a cross-linker modification was assigned
to the lysine at position 216. The respective mass spectra are
shown in Fig. S2 (Supporting Information).
Conclusions and Outlook
The novel thiourea cross-linker described herein is a simplified
analog of our previous reagent incorporating a thiourea-Gly-Pro
structure.[ This cross-linker exhibits a central thiourea moiety,
which is flanked by amine-reactive NHS ester moieties for reaction
with lysines or free N-termini in proteins. The activated ester
moieties exhibit a high reactivity with peptides (substance
P, LHRH) and proteins (hen egg lysozyme and PPARα). The
9]
J. Mass. Spectrom. 2010, 45, 880–891
Copyright ꢀc 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

M. Q. M u¨ ller et al.
Figure 5. (A) MALDI-TOF mass spectrum of nano-HPLC fraction containing a lysozyme interpeptide (type 2) cross-link composed of the N-terminal
+
amino acid connected to amino acids 97–112, in which Lys-1 is cross-linked via its N-terminus with Lys-97 ([M + H] at m/z 2144.023). Other lysozyme
peptides are also visible in the spectrum. (B) CID fragment ion mass spectrum (MALDI-TOF/TOF-MS/MS) of the cross-linked product (m/z 2144.023).
3+
(
C) Nano-ESI-LTQ-Orbitrap-MS/MS data of the cross-linked product [M + 3H] at m/z 715.68. The isolated and fragmented species are represented in
the insets.
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Copyright ꢀc 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 880–891

Fragmentation behavior of a thiourea-based cross-linker
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Copyright ꢀc 2010 John Wiley & Sons, Ltd.
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