Collision-induced dissociative chemical cross-linking reagent for protein structure characterization: Applied Edman chemistry in the gas phase

Article abstract of DOI:10.1002/jms.1702

Chemical cross-linking combined with a subsequent enzymatic digestion andmass spectrometric analysis of the created crosslinked products presents an alternative approach to assess low-resolution protein structures and to gain insight into protein interfaces. In this contribution, we report the design of an innovative cross-linker based on Edman degradation chemistry, which leads to the formation of indicative mass shifted fragment ions and constant neutral losses (CNLs) in electrospray ionization (ESI)-tandem-mass spectrometry (MS/MS) product ion mass spectra, allowing an unambiguous identification of cross-linked peptides. Moreover, the characteristic neutral loss reactions facilitate automated analysis by multiple reaction monitoring suited for high throughput studies with good sensitivity and selectivity. The functioning of the novel cross-linker relies on the presence of a highly nucleophilic sulfur in a thiourea moiety, safeguarding for effective intramolecular attack leading to predictive and preferred cleavage of a glycyl-prolyl amide bond. Our innovative analytical concept and the versatile applicability of the collision-induced dissociative chemical cross-linking reagent are exemplified for substance P, luteinizing hormone releasing hormone LHRH and lysozyme. The novel cross-linker is expected to have a broad range of applications for probing protein tertiary structures and for investigating protein-protein interactions. Copyright

Full text of DOI:10.1002/jms.1702

Research Article
Received: 25 August 2009
Accepted: 1 November 2009
Published online in Wiley Interscience: 30 November 2009
(
www.interscience.com) DOI 10.1002/jms.1702
Collision-induced dissociative chemical
cross-linking reagent for protein structure
characterization: applied Edman chemistry
in the gas phase
a†
b†
b∗
Frank Dreiocker, Mathias Q. M u¨ ller, Andrea Sinz
a∗
and Mathias Sch a¨ fer
Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross-
linked products presents an alternative approach to assess low-resolution protein structures and to gain insight into protein
interfaces. In this contribution, we report the design of an innovative cross-linker based on Edman degradation chemistry,
which leads to the formation of indicative mass shifted fragment ions and constant neutral losses (CNLs) in electrospray
ionization (ESI)-tandem-mass spectrometry (MS/MS) product ion mass spectra, allowing an unambiguous identification of
cross-linked peptides. Moreover, the characteristic neutral loss reactions facilitate automated analysis by multiple reaction
monitoring suited for high throughput studies with good sensitivity and selectivity. The functioning of the novel cross-linker
relies on the presence of a highly nucleophilic sulfur in a thiourea moiety, safeguarding for effective intramolecular attack
leading to predictive and preferred cleavage of a glycyl-prolyl amide bond. Our innovative analytical concept and the versatile
applicability of the collision-induced dissociative chemical cross-linking reagent are exemplified for substance P, luteinizing
hormone releasing hormone LHRH and lysozyme. The novel cross-linker is expected to have a broad range of applications for
Lp tr do .b ing protein tertiary structures and for investigating protein–protein interactions. Copyright ꢀc 2009 John Wiley & Sons,
Keywords: chemical cross-linking; peptides; Edman chemistry; collision-induced dissociation; protein structure analysis
Introduction
we have developed a novel concept for collision-induced dissocia-
tive cross-linking reagents. Thus, our novel cross-linker contains
an extremely labile covalent bond located within the linker region,
which can be selectively and preferably cleaved by low-energy col-
lision activation in the gas phase. For designing this cross-linker,
Chemical cross-linking combined with mass spectrometry (MS) is
a viable approach to study the tertiary and quaternary structure of
proteins and protein complexes.
[
1–3]
However, an unambiguous,
[
23–25]
sensitive,reliableandfastidentificationoftheaminoacidsinvolved
in the covalent derivatization by chemical cross-linking remains
analytically challenging. A mass spectrometric identification of the
chemically modified amino acids in the respective protein is per-
formed after proteolytically digesting the cross-linking reaction
mixtures. This is often hampered by the complexity of the created
peptide mixtures, wherein only a relatively small percentage of
cross-linkedproductsispresentbesidesalargemajorityofunmod-
ified peptides. To safeguard for a selective identification of cross-
wechoseanapproachderivedfromclassicEdmanchemistry
and protein ladder sequencing,[ which has shown to operate
26]
both in the condensed and the gas phase.[
27–30]
Our custom-designed cross-linker leads to the formation of
indicative fingerprint mass shifted product ions and neutral losses
in the product ion mass spectra, which allow an unambiguous
∗
Correspondence to: Andrea Sinz, Department of Pharmaceutical Chemistry
[
4–6]
linked peptides using electrospray ionization (ESI)
combined
& Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-
Wittenberg, Wolfgang-Langenbeck-Straße 4, D-06120 Halle (Saale), Germany.
E-mail: andrea.sinz@pharmazie.uni-halle.de
with collision-induced dissociation (CID) tandem MS (MS/MS),[8]
several approaches have been suggested. They include cross-
linking reagents that incorporate the use of marker ions resulting
[7]
Mathias Sch a¨ fer, Department of Chemistry, Institute of Organic Chem-
istry, University of Cologne, Greinstraße 4, D-50939 Cologne, Germany.
E-mail: mathias.schaefer@uni-koeln.de
[
9,10]
[11]
from low-energy CID
strategies such as proteolytic digestion in 18O-water,[12] isotope
or metastable decay, isotope-coding
[
13–16]
coding of the cross-linking reagents
or isotope coding of the
†
Both authors contributed equally to this work.
[
17]
proteins,
and enrichment of cross-linked products via affinity
Identification of cross-linked peptide ions by MS/MS or
[
18–20]
tags.
a DepartmentofChemistry,InstituteofOrganicChemistry,UniversityofCologne,
Greinstraße 4, D-50939 Cologne, Germany
n
MS is not trivial as product ion spectra of these contain a number
[
21,22]
of product ions, i.e. b- and y-type ions,
peptides involved in the cross-linking product. To enable a more
effective analysis of cross-linked peptides by tandem MS analysis,
originating from both
b DepartmentofPharmaceuticalChemistry&Bioanalytics,InstituteofPharmacy,
Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4,
D-06120 Halle (Saale), Germany
J. Mass. Spectrom. 2010, 45, 178–189
Copyright ꢀc 2009 John Wiley & Sons, Ltd.

Applied Edman chemistry in the gas phase
identification and structure elucidation of cross-linked peptides.
The concept of this cross-linker relies on the presence of a thiourea
moiety (Scheme 1), which is connected to proline via glycine. With
the strong nucleophilicity of sulfur in the thiocarbonyl moiety and
in ethyl acetate. The hydrochloride of 4-amino-butyric acid methyl
ester (2) was isolated by filtration as white solid at a yield of 98%
(Scheme S1 of the Supporting Information).[
43]
For the synthesis of 4-isothiocyanato-butyric acid methyl ester
(3), 2 was suspended in a mixture of dichloromethane and carbon
disulfide. After slowly adding triethylamine, the reaction mixture
was stirred for 30 min, before methyl chloroformate was added.
[
31–33]
the pronounced gas-phase basicity of proline
we combine
[
23–25]
classic Edman degradation chemistry
with the extensive
knowledge on intramolecular peptide fragmentation reactions,[
34]
◦
i.e. the preferred cleavage of peptide bonds besides acidic amino
After refluxing for 4 h, the mixture was cooled to 20 C and poured
[
34–42]
acids as aspartic acid, called the aspartic acid effect.
intowater. Theorganiclayerwaswashedwithdilutedhydrochloric
acid and brine, dried over magnesium sulfate and concentrated
under reduced pressure. The resulting yellowish oil was distilled in
high vacuum to give 3 as colorless oil at a yield of 80% (Scheme S1
of the Supporting Information).[
The proof-of-principle of the presented analytical concept
(Schemes 2–4) and its versatile applicability is demonstrated for
the peptides substance P (amino acid sequence RPKPQQFFGLM-
NH2), luteinizing hormone releasing hormone (LHRH with the
amino acid sequence pEHWSYGLRPG) and hen egg lysozyme
44]
ꢁ
Z-Gly-OSu (5) was synthesized in a N,N -dicyclohexyl carbodi-
(14.3 kDa) as model substances.
imide (DCC)-mediated coupling of benzyloxycarbonyl-glycine
(
4) with N-hydoxysuccinimide (NHS); 4 was suspended in
◦
dichloromethane and NHS and DCC were added at 0 C. After
stirring for 16 h, the solid was removed by filtration and the filtrate
was concentrated under reduced pressure. The residue was stirred
in ethyl acetate and insoluble solid was removed by filtration. After
removal of the solvent under reduced pressure the crude product
Experimental Section
Materials
All chemicals and solvents were used as purchased without further
purification (Acros Organics, Geel, Belgium; ABCR, Karlsruhe,
Germany and Merck KGaA, Darmstadt, Germany). Substance P,
LHRH and hen egg lysozyme were used as purchased from Sigma-
Aldrich, Taufkirchen, Germany. Toluene and tetrahydrofurane
was recrystallized from iso-propanol to give 5 as white solid at
9
ThesynthesisofZ-Gly-Pro-OH(6)startedwithdissolvingsodium
bicarbonate and proline in water. As soon as gas formation had
5% yield (Scheme S1 of the Supporting Information).[
45]
stopped, a solution of 5 in 1,4-dioxane was added. After letting the
(THF) were dried by distillation from benzophenone and sodium,
◦
reaction proceed at 20 C for 1 h, most of the solvent was removed
dichloromethaneandpyridinebydistillationfromcalciumhydride.
Methanol was distilled from magnesia, whereas all other solvents
were distilled over a column prior to use.
under reduced pressure and the residue was acidified to pH 2 with
7
M hydrochloric acid. The solution was extracted three times with
dichloromethane, the combined organic layer were washed twice
with brine, dried over magnesium sulfate, and the solvent was
removed under reduced pressure to give 6 as white solid at a yield
of 90% (Scheme S1 of the Supporting Information).[
Z-Gly-Pro-Gly-OMe (7) was derived from 6 in a DCC-
mediated coupling with glycine methyl ester. 6 was dissolved
Synthesis of the NHS-BuTuGPG-NHS chemical cross-linker
46]
Esterification of 4-amino-butyric acid (1) was achieved by reaction
◦
at 20 C with thionyl chloride in methanol for 15 h. After
evaporation of the reaction mixture, the crude product was stirred
Scheme 1. Structure of the novel homobifunctional chemical cross-linker molecule NHS-BuTuGPG-NHS for structure elucidation of proteins.
Scheme 2. Preferred cleavage of the Gly-Pro amide bond of the linker by selective nucleophilic attack of the sulfur upon CID.
J. Mass. Spectrom. 2010, 45, 178–189
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

F. Dreiocker et al.
Scheme 3. Characteristic fragmentation reaction of intrapeptidal cross-linked peptides with BuTuGPG upon CID, i.e. formation of the CNL of 184 u.
Scheme 4. Characteristic fragmentation reaction of ‘dead-end’ cross-linked peptides upon CID. (a) The nucleophilic attack of the sulfur cleaves off the
Gly-Pro amide bond and generates the five-membered thiazolone, which is lost as BuTuG (CNL of 202 u). (b) The cross-linker is connected via butyric
acid; the analog fragmentation scheme leads to the loss of prolyl-glycin with 172 u.
◦
◦
in dichloromethane, followed by the addition of glycine methyl
ester hydrochloride and triethylamine. DCC was added at 0 C and
0 C. The reaction mixture was brought to 20 C and stirred
for 14 h before it was washed with saturated NaHCO3 solu-
tion and brine. The organic phase was dried over magne-
sium sulfate and the solvent was removed under reduced
pressure. The crude product was purified by column chro-
matography with dichloromethane/methanol (10 : 1 (v/v)) to
give 4-(3-{2-[2-(methoxycarbonylmethyl-carbamoyl)-pyrrolidin-1-
yl]-2-oxo-ethyl}-thioureido)-butyric acid methyl ester (MeO-
BuTuGPG-OMe; 9) as colorless solid at a yield of 77% (Scheme S2
of the Supporting Information).[
◦
the reaction mixture was stirred for 14 h. Urea was removed by
filtration and the filtrate was evaporated. The residue was stirred
in ethyl acetate and insoluble solid was removed by filtration. The
filtrate was washed with 0.2 M NaHCO3 solution, 0.5 M hydrochloric
acid and again with 0.2 M NaHCO3 solution, dried over magnesium
sulfate before the solvent was removed under reduced pressure.
The resulting yellowish oil was purified by column chromatogra-
phy with ethyl acetate/ethanol [20 : 1 (v/v)] to give 7 as colorless
oil at a yield of 73% (Scheme S1 of the Supporting Information).[
Forremovingtheprotectinggroup,7wasdissolvedinmethanol,
treated with palladium on charcoal, and stirred for 16 h under
hydrogen atmosphere (1 bar). After filtration over celite the filtrate
49,50]
47]
Hydrolysis of the two methyl ester functionalities of 9 was
achieved with lithium hydroxide in a mixture of water, methanol,
and THF for 15 hrs. at 20 C. After removing the solvent under
reduced pressure, the precipitate was dissolved in methanol and
the solvent was evaporated. The crude product was purified
by column chromatography with dichloromethane/methanol
(2 : 1 (v/v)) to give dicarboxylic acid 4-(3-{2-[2-(carboxymethyl-
carbamoyl)-pyrrolidin-1-yl]-2-oxo-ethyl}-thioureido)-butyric acid
◦
was evaporated to give H-Gly-Pro-Gly-OMe (8) as gum-like solid at
48]
9
4% yield (Scheme S1 of the Supporting Information).[
The deprotected tripeptide
8
was suspended in
dichloromethane and treated with the thioisocyanate 3 at
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Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 178–189

Applied Edman chemistry in the gas phase
(
HO-BuTuGPG-OH; 10) as colorless solid at 92% yield (Scheme S2
of matrix solution (0.8 mg/ml α-cyano-4-hydroxy cinnamic acid
(HCCA) in 90% ACN, 0.1% TFA, 1 mM NH4H2PO4) and directly pre-
paredontoa384MTP800 µmAnchorChiptarget(BrukerDaltonik).
MALDI-TOF/TOF-MS analyses were conducted in the positive ion-
ization and reflectron mode by accumulating 2000 laser shots in
the range m/z 700–5000 to one mass spectrum. Mass spectra were
externally calibrated using Peptide Calibration Standard II (Bruker
Daltonik). Signals with a signal-to-noise ratio (S/N) >15 were se-
lected for laser-induced fragmentation. In-source decay (ISD) was
performed manually on selected cross-linking product candidates
by increasing the laser energy by ca. 10% relative to the usual laser
energy. 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.
[
51]
of the Supporting Information).
was suspended in pyridine and treated with
N-trifluoroacetoxy succinimide – prepared from NHS and triflu-
oroacetic anhydride – at 0 C. The mixture was brought to
2
1
0
[
52]
◦
◦
0 C within 3 h and pyridine was removed under reduced pres-
sure. The crude product was dissolved in dichloromethane,
washed with 1 M NaHCO3 solution and brine. After dry-
ing the organic phase over magnesium sulfate, toluene was
added and the solvent was removed under reduced pressure
to yield 4-[3-(2-{2-[(2,5-dioxo-pyrrolidin-1-yloxycarbonyl-methyl)-
carbamoyl]-pyrrolidin-1-yl}-2-oxo-ethyl)-thioureido]-butyric acid
2
,5-dioxo-pyrrolidin-1-yl ester (NHS-BuTuGPG-NHS; 11) as col-
orless foam with 86% yield (Scheme S2 of the Supporting
Information).[
53]
Nano-HPLC/nano-ESI-LTQ-Orbitrap-MS
Cross-linking reactions
Fractionation of tryptic peptide mixtures (lysozyme) was carried
outonanultimatenano-HPLCsystem(DionexCorporation,Idstein,
Germany) using reversed phase C18 columns (precolumn: Acclaim
PepMap,300 µm×5 mm,5 µm,100 Å,separationcolumn:Acclaim
PepMap, 75 µm × 150 mm, 5 µm, 100 Å, Dionex Corp., Idstein,
Germany). After washing the peptides on the precolumn for
For cross-linking experiments, aqueous stock solutions of sub-
stance P (100 µg/ml), LHRH (100 µg/ml) or lysozyme (10 mg/ml)
were diluted with 20 mM 4-(2-hydroxyethyl)-piperazine-1-
ethanesulfonic acid (HEPES) buffer (pH 7.4), with 1 ml solution
containing the protein/peptide at a concentration of 10 µM per
aliquot. The cross-linker [200 mM stock solution in dimethylsulfox-
ide (DMSO)] was added in 50-, 100- and 200-molar excess over the
protein/peptide solution and the reactions were allowed to pro-
ceed for 5, 15, 30, 60 and 120 min Reactions were quenched with
ammonium bicarbonate (20 mM final concentration). One 200-µl
1
5 min with water containing 0.1% TFA, they were eluted and
separated using gradients from 0% to 50% B (90 min), 50% to
00% B (1 min), and 100% B (5 min), where solvent A is 5%
1
ACN containing 0.1% formic acid (FA) and solvent B is 80% ACN
containing 0.1% FA. The nano-HPLC system was directly coupled
to the nano-ESI source (Proxeon, Odense, Denmark) of an linear
ion trap LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher
Scientific, Bremen, Germany). MS data were acquired over 122 min
◦
aliquot was taken from each sample and stored at −20 C be-
fore MS analysis. Cross-linked lysozyme was digested with trypsin
◦
(
1 : 100(w/w)enzymetosubstrateratio)overnightat37 Caccord-
[
54]
ing to an existing protocol. The resulting digests were stored at
2
◦
in data-dependent MS mode: Each high-resolution full scan (m/z
−
20 C before MS analysis.
3
00–2000, resolution was set to 60 000) in the orbitrap was
followed by three product ion scans in the orbitrap (resolution
5 000) for the three most intense signals in the full-scan mass
Linear mode MALDI-TOF MS
Matrix-assisted laser desorption/ionization–time-of-flight
1
3
spectrum (isolation window 1.5 u). MS in the orbitrap was
performed on the two most intense signals in the MS spectra
2
(
MALDI-TOF) MS of intact cross-linked lysozyme was performed in
with a resolution of 15 000. Dynamic exclusion (exclusion duration
linear and positive ionization mode on an Ultraflex III instrument
1
80 s, exclusion window −1 to 2 Th) was enabled to allow
(
Bruker Daltonik, Bremen, Germany) equipped with a Smart

detection of less-abundant ions. Data acquisition was controlled
via XCalibur 2.0.7 (ThermoFisher) in combination with DCMS link
beam laser using sinapinic acid (SA) as matrix. 5 pmol of each
sample was applied onto a ground steel target (Bruker Daltonik,
Bremen, Germany) using the double layer method.
2
.0 (Dionex).
Nano-HPLC/MALDI-TOF/TOF-MS
Offline nano-ESI-LTQ-Orbitrap-MS
Tryptic peptide mixtures of lysozyme were analyzed by offline cou-
pling of a nano-HPLC (high performance liquid chromatography)
system (Ultimate 3000, Dionex, Idstein, Germany) to a MALDI-
TOF/TOF mass spectrometer (Ultraflex III, Bruker Daltonik, Bremen,
Germany). 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 separation
column (Acclaim PepMap, C18, 75 µm × 150 mm, 5 µm, 100 Å,
Dionex), which had been equilibrated with 95% solvent A (A: 5%
acetonitrile (ACN), 0.05% TFA). Peptides were separated with a
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 the 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,
http://www.gpmaw.com) and BioTools 3.1.1.36 (Bruker Daltonik,
Bremen, Germany). The length of the cross-linker was calculated
using the Viewer Light 5.0 software (Accelrys).
6
6
0
0-min gradient (0–60 min: 5–50% B, 60–62 min: 50–100% B,
2–67 min: 100% B, 67–72 min: 5% B, with solvent B: 80% ACN,
.04% TFA) at a flow rate of 300 nl/min with UV detection at 214
55]
and280 nm. Eluateswerefractionatedinto18 s. fractionsusingthe
fraction collector Proteineer fc (Bruker Daltonik), mixed with 1.1 µl
J. Mass. Spectrom. 2010, 45, 178–189
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

F. Dreiocker et al.
Results and Discussion
effective condensation with protein amine functionalites.[3] The
actual structure BuTuGPG is a result of the extreme nucleophilicity
of the sulfur in the liquid phase, that lead to the formation
Cross-linker design and rationale
Thestructureofthecross-linkerfeaturesaglycyl-prolylamidebond
that is preferably cleaved by CID. The particular N-terminal bond
adjacent to proline, which is supposed to be substantially more
labile than any other covalentbond in linker-protein condensation
products is predestined for cleavage by an intramolecular attack
of the sulfur of a thiourea moiety (Scheme 2). This fragmentation
reaction results in the formation of a five-membered thiazolone
structure and an N-terminally truncated peptide (here: proline), i.e.
the primary reaction products of the Edman protein degradation
reaction (Scheme 2). The design of the linker is tailored for the
of a number of unwanted cyclized byproducts, i.e. di-keto-
piperazines.[
60–62]
To effectively inhibit these intramolecular
condensation reactions an additional glycine is introduced
C-terminal to proline and the thiourea is connected with butyric
acid. Altogether, these two extra parts of the molecule increase
the length of the cross-linker to 16.7 Å.[
63]
The homobifunctional bisuccinimidyl cross-linker molecule
reacts with amine functionalities, e.g. lysine- and N-terminus
of proteins and peptides.[ Reactivity of NHS-esters toward
3]
functional groups other than amines, e.g. hydroxyl groups in
[
23–25]
[64,65]
fragmentation scheme derived from Edman chemistry
and
serine, threonine or tyrosine is found to be much lower.
After
[
26]
protein ladder sequencing.
The classic Edman degradation
adequate separation of cross-linked proteins with subsequent
proteolytic digestion ESI-MS delivers protonated molecular ions
of cross-linked peptides. Upon low-energy collision activation
(QIT, Triple quadrupole-MS) the Gly–Pro amide bond of the linker
is preferably cleaved by a selective nucleophilic attack of the
sulfur (Scheme 2). The characteristic mass shifted product ions
reaction proceeds via a nucleophilic attack of the sulfur in an
N-terminalphenylthiocarbamyl(PTC)peptidederivative,forminga
five-membered thiazolone structure. Gaskell and others examined
[
27–30]
this reaction in the gas phase,
where collision activated
PTC-peptides give rise to analog thiazolone product ions and
respective peptide fragment ions with the N-terminal amino acid
cleaved off. In consideration of the Edman reaction scheme we
connected a thiourea moiety via glycine to proline, providing the
prerequisites that the Gly-Pro amide bond is substantially weaker
than any other covalent bond in either the attached peptides or
the linker itself (Scheme 2). This can be assumed as the sulfur in
the thiocarbonyl group is a much more effective nucleophile than
n
are further examined by MS -experiments, in which the primary
structure of the peptides and localization of the linker is reliably
elucidated by indicative neutral losses as shown for the model
peptide substance P in Schemes 3 and 4. This feature is important
to improve the capabilities of the cross-linking approach, since
characteristic secondary fragmentation reactions allow an elegant
identification of chemical cross-linking products with improved
sensitivity and selectivity and provide a straightforward access
to automated detection by multiple-reaction monitoring (MRM).
Mass measurement of the intact cross-linked species, followed by
[
27,56]
any carbonyl oxygen.
Moreover, the very basic amino acid
proline (gas-phase basicity: 213.3 kcal/mol[31–33]) is placed in the
linker to promote the initial protonation at its amide nitrogen.
The protonation at this site is reasonable, especially in multiply-
protonated molecular ions of proteins or peptides. In very basic
peptides or proteins, in which preferably even more basic amino
acids such as Arg and Lys are protonated, a proton shift to the
proline in the linker can be assumed to take place upon collision
n
MS analysis of each of the peptide chains, will provide both the
identities of cross-linked proteins and information regarding the
sites of derivatization.
Cross-linking with substance P
[
41,57]
activation according to the mobile proton model.
However, the functioning of our Edman chemistry-based
collision-induceddissociativechemicalcross-linking reagentrelies
on the correct understanding of intramolecular peptide fragmen-
tation reactions, which are still a matter of scientific debate.
Recently, the current status of knowledge on intramolecular pep-
tide fragmentation reactions was exhaustively reviewed by Paizs
and Suhai.[ Especially, the collision-induced cleavage of the
Asp–Pro amide bond is extensively examined, for which a charge
remote fragmentation mechanism is strongly favored. That reac-
tionschemestartswiththeprotontransferfromthecarboxylicacid
To demonstrate the functioning of the analytical concept
substance P was chosen as a model peptide that was treated
withNHS-BuTuGPG-NHS. Thepositiveelectrospraymassspectrum
of the reaction products is shown in Fig. 1(a). As characteristic
fragment ions evidence, the N-terminus of substance P and
the side chain amino group of the lysine at position 3 are
connected by the cross-linker BuTuGPG. The doubly protonated
molecular ion of the intramolecularly cross-linked substance P
with BuTuGPG is found at m/z 843.42, the singly charged analog
is observed at m/z 1685.84. Additionally, two reaction products
of NHS-BuTuGPG-NHS with substance P are found, in which the
cross-linker is partially hydrolyzed and hence only connected
via a single amide bond. These ‘dead-end’ cross-linked ions are
found with one (at m/z 852.43) and with two linker molecules
34]
of Asp to the very basic backbone amide nitrogen of the adjacent
prolyl residue.[
35,39,40]
This delivers an intermediate zwitterion,
which in turn promotes the nucleophilic attack of the carboxylate
anion of the aspartyl side chain to the neighbored carbonyl car-
bon. The intramolecular reaction finally leads to the formation of
(at m/z 1032.97) attached to substance P.[ The MS product
66]
2
a cyclic anhydride of the aspartyl residue and cleaves off the pro-
ion spectrum of the intramolecular cross-linked precursor ion
[M+BuTuGPG + 2H] at m/z 843.42 is presented in Fig. 1(b).
line N-terminally.[
35,39–42]
Based on the experimental observation
2+
that the Asp–Pro bond is easily cleaved and the advanced un-
derstanding of the mechanism, Soderblom and Goshe presented
an aspartyl–prolyl moiety in a chemical cross-linker to selectively
Obviously, the vulnerable Gly–Pro bond in the cross-linker is
preferably cleaved upon CID. A product ion at m/z 751.41 is
released, which is formed by the characteristic loss of 184 u.
A proposed mechanism for the generation of the neutral loss
expelled is shown in Scheme 3. The nucleophilic attack of the
sulfur cleaves off the Gly-Pro amide bond and generates the five-
membered thiazolone. In the depicted reaction the prolyl moiety
is protonated. Subsequently, the thiazolone rearranges and loses a
neutral fragment with 184 u either via a charge remote (depicted)
[
58,59]
dissociate this particular bond upon collision activation.
The abbreviation BuTuGPG for the novel linker used throughout
the article shown in Scheme 1 is deduced from the respective
one letter codes of the amino acids glycine (G) and proline
(P) and from thiourea (Tu) and amino-butyric acid (Bu). N-
hydroxysuccinimidyl active ester is placed on both ends to enable
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Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 178–189

Applied Edman chemistry in the gas phase
2
2+
Figure 1. (a) ESI-mass spectrum of substance P modified with BuTuGPG; (b) MS -CID-product ion spectrum of [M+BuTuGPG + 2H] at m/z 843.42
∗
acquired in a linear ion trap, fragment ions were analyzed in the orbitrap. The signal marked with an asterisk ( ) belongs to an additional fragmentation
3
2+
2
of the thiourea group; (c) MS -CID-product ion spectrum of [M+PG + 2H] at m/z 751.41; (d) MS -CID-product ion spectrum of [M+Linker−OH +
2
+
∗
2
H] at m/z 852.43 acquired in a linear ion trap, fragment ions analyzed in the orbitrap. The signals marked with an asterisk ( ) belong to an additional
fragmentation of the thiourea group.
n
or via a charge driven mechanism. Accurate mass measurements
of precursor and product ion allowed the determination of the
composition of this neutral fragment to be C7H8N2SO2, for which
we propose a cyclized BuTuG structure. The fingerprint loss of 184
uisthereforeideallysuitedforselectiveandsensitiveidentification
of intra- and intermolecular cross-linked peptides with our new
linker. Hence, peptides decorated with the BuTuGPG linker can
be tracked down by a CNL experiment for 184 u. After reliable
identificationofcross-linkedpeptidesbythisCNLscan,theprimary
structure of the modified peptides is determined by successive
MS product ion experiments. This strategy is demonstrated for
2
respective ions of substance P at m/z 843.42 (MS ; Fig. 1(b)) and
m/z 751.41 (MS ; Fig. 1(c)). On the basis of the mass shift of
3
either the complete linker (ꢀm = 338 u), here connecting the
N-terminus with lysine at position 3, or the linker remains on the
peptide after CID (Pro-Gly moiety: ꢀm = 154 u), the location of
the covalent modification can be deduced straightforwardly. The
almost complete series of b-fragment ions and additional y-ions
[
43]
n
observed in the MS spectra allow a reliable assignment (Fig. 1(b)
and (c)).
J. Mass. Spectrom. 2010, 45, 178–189
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

F. Dreiocker et al.
Figure 1. (continued).
It is important to note that the signal at m/z 843.42 (Fig. 1(a))
assigned to the doubly protonated molecular ion of intrapeptidal
cross-linked substance P could also originate from an isobaric ion
of substance P modified with a dehydrated, singly condensed
moiety. Moreover, all cyclized byproducts found during the linker
synthesis in the condensed phase are exclusively generated by
intramolecular nucleophilic reactions of the sulfur (see preceding
section). Yet, an involvement of the sulfur in the water loss
discussed here can be excluded, as the thiocarbonyl sulfur is
indispensable for the subsequent formation of the characteristic
neutral loss, i.e. 184 u (mechanism A in Scheme 4).
Especially the formation of the y10- and y10-NH3 fragment
ions observed in Fig. 1(b) has to be discussed in the light of
these considerations. In the intrapeptidal cross-linking product
of substance P the butyric acid of the BuTuGPG linker can be
connected either to lysine 3 (see structure B in Fig. 1(b)) or to
‘dead-end’ cross-linker. For the formation of the latter only the
free butyric acid part of a partially hydrolyzed cross-linker (see
structure A shown in Fig. 1(d)) is considered to be suited for
cyclization accompanied with a loss of a water molecule (18 u). The
intramolecular condensation reaction is entropically demanding
and is somewhat unlikely as the thiourea amide nitrogen with
only moderate nucleophilic character has to be responsible for
the formation of the δ-lactame derived from the butyric acid
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Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 178–189

Applied Edman chemistry in the gas phase
the N-terminus (see structure A in Fig. 1(b)). Both isomers deliver
a characteristic loss of 184 u upon CID (Scheme 3), but only the
former of the modified peptides (structure B in Fig. 1(b)) can give
rise to y10- and y10-NH3 ions withouta respective mass shiftrelated
to the linker (see structure B in Fig. 1(c)). Given a dehydrated dead-
end linker with a cyclized butyric acid δ-lactame connected to
the N-terminus is present, a neutral loss of 184 u could also be
formed by this ion, as the characteristic neutral loss of 202 u
OH) is lost (CNL of 172 u). The fact that both CNLs are observed
in the product ion spectrum (Fig. 1(d)) of the precursor ion at m/z
852.43 can straightforwardly be explained by the presence of both
isobaric ‘dead-end’ linked peptides depicted (structures A and B in
Fig. 1(d)). The fragment ions observed in the MS spectrum allow
locating the chemical derivatization to Lys-3 of substance P.
3
n
Cross-linking with LHRH
would be shifted by 18 u (H2O). Hence, from this MS -data alone
it cannot be excluded that the y10- and y10-NH3 ions could also
be formed by respective fragmentation reactions of an isobaric
peptide precursor modified with a dehydrated dead-end linker.
In addition to substance P, LHRH was chosen as another
model peptide to investigate the properties of the novel
cross-linker. ESI-LTQ-Orbitrap-MS and MS/MS data (Figs S1 and
S2 of the Supporting Information) were collected confirming
the fragmentation behavior that had already been observed
for substance P. As the primary structure of LHRH offers no
possibility for an intramolecular cross-link, only dead-end cross-
linked product ions are found. Tellingly, the two characteristic
CNL fragments of 172 and 202 u confirm the presence of both
possible dead-end linked isomers (Fig. S2 of the Supporting
Information). Moreover, in Fig. S2 of the Supporting Information
the dead-end linked peptides of LHRH do not show any water loss
(ꢀm = 18 u) upon CID. This experimental result further supports
the assumption that the water loss proceeds via a complicated
rearrangement reaction, i.e. the formation of the δ-lactame from
linear butyric acid, which is not favored in the gas phase upon CID,
making the formation of respective dehydrated species unlikely.
However, we note that a constant neutral loss (CNL) of 154 u
2
(
172 u − 18 u = 154 u) is absent in all MS product ion spectra
of assigned intramolecular cross-links as shown in Figs 1(b), 3(a)
and 4(a), showing that a dehydrated dead-end linked peptide
connected via the butyric acid is completely missing. This is
reasonable as a formation of a β-lactame as result of a C-terminal
water loss of glycine is at least sterically strongly hindered.
2
In Fig. 1(d), the CID MS product ion spectrum of substance P
modified with the partially hydrolyzed cross-linker is presented.
For this ‘dead-end’ cross-link peptide modification two indicative
neutral losses of either 172 or 202 u are observed upon CID.[ As
illustrated in Scheme 4, these two CNL reactions are characteristic
for the directivity of linkage as either the thiazolone-butyric acid
43]
(
HO-BuTuG) is eliminated (CNL of 202 u) or the prolyl-glycin (PG-
·
50 Cross-linker
·
100 Cross-linker
200 Cross-linker
·
1
4313
14313
14313
1
00
Lysozyme
5
0
14478
1
4478
14478
2
3
4
5
3
4
5
6
4
5
6
0
1
5678
15682
16033
1
5340
1
1
00
15681
1
6029
1
5339
5
0
0
14993
1
16379
16379
1
6017
5 min
1
5677
5677
5677
15682
16032
5677
00
16029
1
5338
5
0
1
6016
15 min
0
1
15678
15680
16026
5680
1
1
1
00
16032
16371
16025
16374
1
1
5337
5
0
0
16017
16023
16381
3
6
1
0 min
0 min
20 min
1
16022
5682
00
1
5
0
0
15342
1
6378
16022
1
5678
16030
00
15679
1
5682
1
5341
16378
5
0
0
16015
16372
1
4000
15000
16000
14000
15000
m/z
16000
14000
15000
m/z
16000
m/z
Figure 2. Linear MALDI-TOF-MS of lysozyme; excess of Edman cross-linker (50-, 100- and 200-fold excess) and reaction times (5–120 min) are indicated.
The calculated average molecular weight of hen egg lysozyme is 14 313 Da matching perfectly with the signal at m/z 14 313. The signal at m/z 14 478
probably corresponds to a glycosylated species of lysozyme.
J. Mass. Spectrom. 2010, 45, 178–189
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

F. Dreiocker et al.
2
Figure 3. Chemicalcross-linkingdata analysis forlysozyme peptide (amino acids 6–14), modified with the BuTuGPG cross-linker. (a) ESI-LTQ-Orbitrap-MS
product ion spectrum of the precursor ion [M+BuTuGPG + 2H] at m/z 694.15 and (b) ESI-LTQ-Orbitrap-MS product ion spectrum of [M+PG + 2H]
at m/z 602.43.
2+
3
2+
Cross-linking with lysozyme
In Fig. 3(b) and 3(b), chemical cross-linking data analysis is
presented exemplarily for lysozyme peptide (amino acids 6–14),
being modified with the BuTuGPG cross-linker. Product ions
resultingfromuptothreewaterlossesarevisibleintheproduction
spectra of derivatized lysozyme peptides (Fig. 3(a)). The multiple
loss of water rather originates from unspecific fragmentations of
the lysozyme peptide (amino acids 6–14) than from the linker
molecule alone. MALDI-MS/MS data confirm the observation that
one water molecule is easily lost from the peptide modified with
a partially hydrolyzed cross-linker, probably already during the
cross-linking reaction (data not shown). The primary fragment ion
Inordertoevaluatetheapplicabilityofourchemicalcross-linkerfor
proteins the 14.3 kDa protein lysozyme was derivatized with the
novel collision-induced dissociative reagent. Efficientcross-linking
was proven by MALDI-TOF mass spectrometric measurements in
the linear mode showing the attachment of up to six cross-
linker molecules to the protein depending on molar excess
(50-, 100- and 200-fold excess) of cross-linker and reaction time
(5–120 min) (Fig. 2). Modified lysozyme was in-solution digested
with trypsin and the resulting peptide mixtures were analyzed by
both nano-HPLC/MALDI-TOF/TOF-MS and nano-HPLC/nano-ESI-
LTQ-Orbitrap-MS.
2
+
2
[M+PG + 2H] at m/z 602.43 generated in a MS experiment by
3
the loss of 184 u was subjected to a MS product ion experiment
www.interscience.wiley.com/journal/jms
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 178–189

Applied Edman chemistry in the gas phase
2
2+
Figure 4. (a) ESI-LTQ-Orbitrap-MS product ion spectrum of [M+BuTuGPG + 2H] of intrapeptidal cross-linked (Lys-116 to Thr-118) lysozyme peptide
3
2+
(
amino acids 115–125) at m/z 836.39 and (b) ESI-LTQ-Orbitrap-MS product ion spectrum of the precursor ion [M+PG + 2H] at m/z 744.57 formed by
3
CID in a ESI-LTQ-MS experiment.
2
by CID in the LTQ yielding an almost complete y-type ion series of
the modified lysozyme peptide (Fig. 3(a)).
The precursor ion at m/z 836.39 was further analyzed in an MS
2
+
experiment (Fig. 4(a)). The [M+PG + 2H] ion at m/z 744.57 is
formed by the characteristic CNL of 184 u (loss of BuTuG). For
the same reasons as discussed for substance P we assume that
the precursor ion at m/z 836.39 is representing the intrapeptidal
cross-linked product of the lysozyme peptide containing amino
acids 115–125.
An obvious drawback of the presented chemical cross-linker
with its pronounced ability for collision-induced cleavage is
certainly its sophisticated synthesis, which is time consuming
and therefore costly. In addition, the elimination of remaining
An intriguing case is presented by lysozyme peptide comprising
amino acids 115–125 (Fig. 4(a)). In the respective ESI-LTQ-Orbitrap
mass spectrum a signal at m/z 836.39 was detected, formally
corresponding to the doubly charged ion of an intrapeptidal
cross-linked species. For this species, the only reactive site in
addition to Lys-116 would be the threonine residue at position
1
18. It has been shown that NHS cross-linkers can also react with
serines, threonines and tyrosines in addition to lysines and free
[
64,65]
N-termini, albeit with lower frequency.
J. Mass. Spectrom. 2010, 45, 178–189
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

F. Dreiocker et al.
ambiguities regarding the formation of dehydrated and partially
hydrolyzed cross-linker peptide derivatives besides intrapeptidal
cross-linked products motivated us to further refine this approach
and to develop a second generation thiourea linker with
advanced characteristics. The properties of a simplified cross-
linkeranalogueoftheNHS-BuTuGPG-NHScompoundarecurrently
under investigation.
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Electrospray ionization for mass spectrometry of large
biomolecules. Science 1989, 246, 64.
[
5] R. Cole. Electrospray Ionization Mass Spectrometry: Fundamentals,
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7] J. M. Wells, S. A. McLuckey. The Encyclopedia of Mass Spectrometry,
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Protein sequencing by tandem mass spectrometry. Proc. Nat. Acad.
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The proposed dissociative chemical cross-linker incorporates a
thiourea moiety, which contains highly nucleophilic sulfur for
respectiveattackattheadjacentGly-Proamidecarbonyl,efficiently
initiating the cleavage of the linker molecule. The underlying
reaction mechanism was examined and the resulting mass shifts
and neutral losses were found to provide reliable information on
the connectivity of the linker and the location of the respective
covalent derivatization. This fundamental study shows that the
concept of this dissociative chemical cross-linker has the potential
to improve data evaluation of cross-linked proteins and peptides
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[
3
by detailed MS/MS and MS studies. However, the synthesis of
[
12] J. W. Back, V. Notenboom, L. J. de Koning, A. O. Muijsers, T. K. Sixma,
the presented cross-linker proved to be more challenging than
expected. Therefore, we extended the thiourea-based concept for
a dissociative cross-linker to a simplified analog, which is currently
under investigation in our laboratories.
C. G. de Koster, L. de Jong. Identification of cross-linked peptides
18
for protein interaction studies using mass spectrometry and
O
labeling. Anal. Chem. 2002, 74, 4417.
[
[
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Proteins 2007, 69, 254.
The novel dissociative chemical cross-linker safeguards for an
effective cleavage in the low (5–100 eV) as well as in the high-
energyregime(keV)ofCIDexperiments.Thespecialfragmentation
of the linker molecule delivers indicative mass shifts and CNLs,
enabling sensitive, selective and unambiguous detection by MRM.
Moreover, our approach allows a facile automation for multi-
sample unequivocal identification of cross-linked peptides even
without the need for high accuracy mass analyzers (e.g. quadruple
ion traps and triple quadrupole mass analyzers) as highly selective
CNLs are utilized for the identification of cross-linked species. With
all requirements of this complex analytical problem addressed the
developed technique should raise interest to those who study
protein–protein interactions using chemical cross-linking and
have access to any mass spectrometer capable of MS/MS analysis.
[
15] C. Ihling, A. Schmidt, S. Kalkhof, C. Stingl, K. Mechtler, M. Haack,
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[
[
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[
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[
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