Quaternary diamines as mass spectrometry cleavable crosslinkers for protein interactions

Article abstract of DOI:10.1007/s13361-011-0288-4

Mapping protein interactions and their dynamics is crucial to defining physiologic states, building effective models for understanding cell function, and to allow more effective targeting of new drugs. Crosslinking studies can estimate the proximity of proteins, determine sites of protein-protein interactions, and have the potential to provide a snapshot of dynamic interactions by covalently locking them in place for analysis. Several major challenges are associated with the use of crosslinkers in mass spectrometry, particularly in complex mixtures. We describe the synthesis and characterization of a MS-cleavable crosslinker containing cyclic amines, which address some of these challenges. The DC4 crosslinker contains two intrinsic positive charges, which allow crosslinked peptides to fragment into their component peptides by collision-induced dissociation (CID) or in-source decay. Initial fragmentation events result in cleavage on either side of the positive charges so crosslinked peptides are identified as pairs of ions separated by defined masses. The structures of the component peptides can then be robustly determined by MS ³ because their fragmentation products rearrange to generate a mobile proton. The DC4 crosslinking reagent is stable to storage, highly reactive, highly soluble (1 M solutions), quite labile to CID, and MS³ results in productive backbone fragmentation.

Full text of DOI:10.1007/s13361-011-0288-4

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2012) 23:201Y212
DOI: 10.1007/s13361-011-0288-4
RESEARCH ARTICLE
Quaternary Diamines as Mass Spectrometry
Cleavable Crosslinkers for Protein Interactions
1
2
1,3,4
Billy Clifford-Nunn, H. D. Hollis Showalter, Philip C. Andrews
1
Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI( USA
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan Vahlteich Medicinal Chemistry Core,
2
Ann Arbor, MI( USA
3
Department of Biological Chemistry, University of Michigan Medical School, Room 1198, 300 North Ingalls Building, 300
North Ingalls St., Ann Arbor, MI 48109, USA
4
Center for Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI( USA
Abstract
Mapping protein interactions and their dynamics is crucial to defining physiologic states, building
effective models for understanding cell function, and to allow more effective targeting of new
drugs. Crosslinking studies can estimate the proximity of proteins, determine sites of protein–
protein interactions, and have the potential to provide a snapshot of dynamic interactions by
covalently locking them in place for analysis. Several major challenges are associated with the
use of crosslinkers in mass spectrometry, particularly in complex mixtures. We describe the
synthesis and characterization of a MS-cleavable crosslinker containing cyclic amines, which
address some of these challenges. The DC4 crosslinker contains two intrinsic positive charges,
which allow crosslinked peptides to fragment into their component peptides by collision-induced
dissociation (CID) or in-source decay. Initial fragmentation events result in cleavage on either
side of the positive charges so crosslinked peptides are identified as pairs of ions separated by
defined masses. The structures of the component peptides can then be robustly determined by
3
MS because their fragmentation products rearrange to generate a mobile proton. The DC4
crosslinking reagent is stable to storage, highly reactive, highly soluble (1 M solutions), quite
3
labile to CID, and MS results in productive backbone fragmentation.
Key words: Mass spectrometry, Crosslinking, Aldolase, Subunit interactions, Protein crosslinker
purify complexes and then identify their components using
mass spectrometry. Hydrogen–deuterium exchange, timed
Introduction
ass spectrometry-based methods have been extensively
used to characterize protein structures and their inter-
actions due to their sensitivity, speed, and accuracy [1–7].
Purification techniques such as tandem affinity purification
TAP) tagging and affinity purification have been used to first
oxidation, and limited proteolysis, in combination with mass
spectrometry, as well as mass spectrometry of intact complexes
are methods used for evaluation of protein secondary, tertiary,
and quaternary structures [1–7]. Each of these methods has
limitations when applied to global analysis of protein inter-
actions, whether it is in the relatively lower throughput of
hydrogen–deuterium exchange, the loss of weakly associated
proteins during TAP tagging, and other methods that require
isolation of protein complexes or lower sensitivities for intact
protein analyses.
M
(
Electronic supplementary material The online version of this article
doi:10.1007/s13361-011-0288-4) contains supplementary material, which
is available to authorized users.
(
Chemical crosslinking combined with mass spectrometry
can provide complementary information to these methods
Correspondence to: Philip C. Andrews; e-mail: andrewsp@umich.edu
Received: 29 August 2011
Revised: 20 October 2011
Accepted: 22 October 2011
Published online: 1 December 2011

2
02
B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
and has several potential advantages [8]. The weaker binding
partners that might be lost during affinity isolation are not
lost during chemical crosslinking because crosslinking locks
protein interactions in place [9]. Interacting partners can be
determined, spatial information can be obtained, relatively
small quantities of protein are required, and data can be
generated from crosslinking experiments more rapidly than
some other methods.
search groups. Properties of these new crosslinkers include a
broader range of chemical reactivities, improved fragmenta-
tion properties in MS/MS, and the addition of affinity
ligands [18–30]. It is worth noting that most of these affinity
methods also target the dead-end reaction products, which
are in large excess over the type 2 crosslinked peptides and
thus the informative peptides still constitute a minor
population of the enriched peptides. In addition, many
crosslinking reagents are not water soluble or have limited
solubility. The organic solvents required to solubilize these
crosslinkers can directly affect protein interactions and
structures, leading to experimental artifacts.
Identification of the rare crosslinked peptides has also
been facilitated by use of a mixture of heavy and light
isotopically labeled crosslinking reagents and chemical tags
[31]. Mass analysis of the peptides identifies ions separated
by the distinctive mass corresponding to the number of
hydrogen atoms substituted by deuterium [9, 32–34]. Dead-
end peptides also produce pairs of ions and computational
schemes have been developed which filter these spectra out
as part of the identification process [35–38]. Crosslinked
peptides (type 2) that produce marker ions upon fragmenta-
tion facilitate the identification of the spectra and to our
knowledge few crosslinkers have been identified to do so,
specifically with type 2 peptides [14, 39].
A number of challenges have been traditionally associated
with mass spectrometric analysis of crosslinked peptides
[
9–12]. Crosslinked peptides (type 2 crosslinks – see
Table 1) in a proteolytic digest are generally very low in
abundance relative to noncrosslinked peptides. Sample
complexity is increased by the presence of side reactions,
intracrosslinked peptides (type 1) and dead-end reactions
[
type 0 in which one end of the crosslinker reacts with
protein and the other end with a nucleophile (e.g., water)] [13,
4]. Tandem mass spectra of crosslinked peptides often
1
undergo diminished fragmentation relative to linear peptides
by collision-induced dissociation or are inherently complex
due to overlapping b- and y-ion series from both peptides
[12, 15].
The nature of the crosslinking chemistry can also
introduce challenges. For example, N-hydroxysuccinimide
ester is a common reactive group used to crosslink proteins
and reacts primarily with free amines such as the epsilon
nitrogen in lysyl residues. There is evidence that seryl,
threonyl, and tyrosyl residues react as well [16, 17], but
appropriate data analysis algorithms can take advantage of
these extended specificities to potentially identify additional
sites of crosslinking. In addition, proteins crosslinked at
lysyl residues have fewer sites available for tryptic digestion,
resulting in larger peptides. These larger peptides may not
fragment as effectively by collision-induced dissociation
To overcome the poor fragmentation properties of cross-
linked peptides, both chemically cleavable and mass
spectrometry cleavable crosslinking reagents have been
developed [40–43]. Chemical cleavage of the crosslinked
peptides prior to mass spectrometry leads to ambiguity
regarding which two cleaved peptides were crosslinked,
particularly in complex mixtures. More desirable are cross-
linkers that fragment efficiently in MS/MS mode to yield
two major fragment ions corresponding to the component
3
(
CID) around the crosslinked site and may have complex
peptides which can subsequently be subjected to MS for
fragmentation patterns making the spectra difficult to
identify and interpret. To overcome these obstacles, several
crosslinking reagents are commercially available and new
crosslinkers are being actively developed by various re-
identification. Thus, MS/MS provides a clear precursor/
product relationship indicating unambiguously that the two
product peptides were covalently crosslinked. Several recent
efforts have addressed the design of these MS-cleavable
crosslinking reagents, leading to new crosslinkers with
improved functionality [15, 22, 44–51], including electron
transfer dissociation (ETD) cleavable reagents [30, 52] but
further improvements are still needed.
Balancing the properties of the reagents to obtain good
reactivity in solution and stability in MS mode while
maximizing fragmentation of the crosslinker for a variety
of peptides in MS/MS mode is challenging. Many of the
crosslinking studies use model proteins but publications
analyzing crosslinks of more complex systems are only
recently starting to appear, in part because of computational
limitations in data analysis and also because reagents that
work for model peptides do not necessarily work well for the
diverse peptides derived from intact proteins [26, 53].
We have completed the synthesis and initial charac-
terization of a new crosslinking reagent DC4, (Figure 1)
based on cyclic diamine structures highly susceptible to
Table 1. During a Crosslinking Reaction, Three Types of Modifications
can Occur, Resulting in Three Types of Peptide Products. Dead-End
Products Result when the Crosslinking Reagent Reacts on One End with
Protein and Does Not Couple on the Other End or Reacts with a Small
Molecule (e.g., Water) on the Other End. Loop Links Occur When Both
Ends of the Crosslinking Reagent React within the Protein but No
Proteolytic Cleavage Occurs between the Two Sites. When Proteolytic
Cleavage Occurs between the Two Sites or the Sites are on Different
Proteins, a Crosslinked Peptide is the Result

B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
203
Synthesis
2
1
,5-Dioxopyrrolidin-1-yl 4-bromobutanoate (1): Compound
was prepared as previously described by Almirante et al.
with minor modifications [54]. Briefly, a mixture of 4-
bromobutyric acid (24 g, 144 mmol), N-hydroxysuccinimide
(19.8 g, 172 mmol), N,N-dicyclohexylcarbodiimide (35.5 g,
1
72 mmol) 4-(dimethylamino)pyridine (3.5 g, 28.6 mmol),
and dichloromethane (300 mL) was stirred overnight at
room temperature. Precipitated N,N-dicyclohexylurea was
filtered off, washed with dichloromethane, and the filtrate
was concentrated to an oil that was purified by flash silica
gel chromatography eluting with 30:70 ethyl acetate:hex-
anes. Approximately 200 mL fractions were collected and
Figure 1. The crosslinker synthesized was named DC4 and
spans approximately 18Å. The 1,4-diazabicyclo[2.2.2]octane
moiety within DC4 is abbreviated as DABCO and will be
referred to as such throughout this paper as such
1
each was concentrated and assayed by H NMR. Fractions
fragmentation and having a number of other desirable
properties. The DC4 crosslinker has been characterized
with model peptides to determine its fragmentation
behavior in both electrospray ionization (ESI) and
matrix-assisted laser desorption ionization (MALDI) and
then validated using the multimeric model protein
aldolase. Good efficiencies of fragmentation were observed
with both ionization methods. Fragmentation occurs within the
crosslinking moiety adjacent to both nitrogen atoms, producing
two intact peptide fragments that can be further fragmented,
yielding a robust series of b- and y-ions for peptide
identification.
containing pure product were combined to give 3 (23.3 g,
1
61.4 %) as a white solid: mp 58–64 °C; H NMR (d
DMSO)
6
1
3
δ 3.6 (t, 2H), 2.8 (m, 6H), 2.2 (m, 2H);
(d DMSO) δ 170.64 (C=O), 168.64 (C=O), 33.38 (CH
29.47 (CH ), 27.97 (CH ), 25.92 (CH ).
C NMR
Br),
6
2
2
2
2
1,4-Bis(4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-
1,4-diazabicyclo[2.2.2]octane-1,4-diium dibromide (2): A
mixture of NHS ester (1) (2 g, 7.5 mmol), 1,4-
diazabicyclo[2.2.2]octane (DABCO; 403 mg, 3.6 mmol)
, and DMSO (6 mL) was stirred at room temperature for
1 wk. Acetonitrile was added to precipitate the product,
which was collected by filtration, washed with acetoni-
trile, and dried to yield (2) (1.7 g, 73.6 %) as a white
1
solid: mp 230 °C (dec); H NMR (d DMSO) δ 3.9 (s,
6
6
1
3
H), 3.6 (t, 4H), 2.8 (m, 12H), 2.1 (m, 4H); C NMR
Experimental
(
d DMSO ) δ 170.64 (C=O), 168.64 (C=O), 62.4 (CH ),
6
2
Materials
51.08 (CH
MS (ES ) m/z 559.2, 561.2 (MBr) .
2
), 27.42 (CH
2
), 25.94 (CH
2
), 17.55 (CH
2
);
+
+
N-terminally acetylated peptides Ac-QIGKGVAR, Ac-
QHAKLGR, were synthesized by the Biomedical Research
Core Facility at the University of Michigan Medical School
using standard Fmoc-based methods, a PTI Symphony synthe-
sizer and purification by reversed-phase HPLC. All peptides
were at least 90% pure and had the expected intact masses and
fragmentation patterns. Trypsin was TPCK treated from
Worthington Biochemical Corporation. Deuterated solvents
were purchased from Cambridge Isotope (Andover, MA,
USA). Rabbit aldolase and all other reagents were purchased
from Sigma Aldrich or Fisher Scientific and used without
further purification.
Crosslinking Reaction
In all experiments, 100 mM crosslinker was prepared
immediately before use in 250 mM HEPES, pH 7.0. The
acetylated standard peptides (10 ug of a 1 ug/uL solution)
were reacted overnight with a 5 molar ratio of crosslinker.
Fifty micrograms of a freshly prepared 5 μg/μL solution of
aldolase was crosslinked using a 25-fold molar ratio of
crosslinker to lysine residues for 30 min.
Mass Spectrometry
Prior to mass spectrometry, the crosslinked synthetic
peptides were dried using a vacuum centrifuge. The dry
peptides were reconstituted in 0.1% trifluoroacetic acid and
Millipore C18 ZipTips were used to desalt the fractions
according to the manufacturer’s instructions. The pH of the
crosslinked aldolase was adjusted to 8.0 with 100 mM
triethylammonium bicarbonate and then the solution was
digested with trypsin in a 1:20 substrate to enzyme ratio
overnight at 37 °C. The tryptic digest was further digested
with endoprotease GluC (50:1) overnight at 37 °C. An
Instrumentation for Crosslinker Synthesis
Melting points were determined in open capillary tubes on a
Laboratory Devices Mel-Temp apparatus and are uncorrected.
The NMR spectra were recorded on a Bruker instrument at
1
13
5
00 MHz for H and 125 MHz for C spectra. Chemical shift
values are recorded in δ units (ppm). Mass spectra of the
synthesized compounds were recorded on a Micromass LCT
time-of-flight mass spectrometer with an electrospray ioniza-
tion source.

2
04
B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
HPLC equipped with a Gilson 811 C dynamic mixer, Gilson
07 and 305 pumps, Scientific Systems model CP-21 Lo
Identification of Crosslinked Peptides
with a Custom Algorithm and Database
Searching Software
3
Pulse; Amersham Biosystems 759A absorbance detector set
at 214 nm, and Isco Foxy 200 fraction collector was used
with a C18 PLRP-S 5 μ 100Å column to separate the
peptides. Solvent A was 5% acetonitrile, 0.1% trifluoroacetic
acid (TFA) while Solvent B was 90% acetonitrile 0.1%
TFA. The flow rate was set to 1 mL/min with the following
binary gradient: 0 min, 6.5% B, 14 min, 6.5% B, 17 min,
A custom database containing all standard proteins used in
our lab was searched using the Mascot search engine. The
searches were completed using a peptide mass tolerance of
0.5 Da, fragment mass tolerance of 0.6 Da, and no enzyme.
Variable modifications included the deamidation of gluta-
mine and asparagine residues, oxidation of methionine,
pyro-Glu, and the modification of lysine residues or the N-
terminus that results from in source decay (in-source decay)
of the crosslinker (+68 Da).
Algorithms written in Java were used to perform three filters
of the observed masses to identify potentially modified
peptides according to Scheme 1. Peak lists from the MS mode
were extracted from the ABI 4800 TOF/TOF. A 0.5 Da mass
tolerance was allowed for the searches. The software first
searched for differences of 112 Da between two peaks. In the
second filter, candidate dead-end peptides were identified by
searching for a set of three peaks: a precursor mass, a loss of
1
8% B, 62.4 min, 40%B, 77.8 min, 50% B, 83.4 min, 70%
B, 84.8 min, 100% B, 86.2 min, 100% B, 87.6 min, 6.5% B,
6 min, 6.5% B. One minute fractions were collected and
9
then dried using a Jouan speedvap. Dried fractions were
reconstituted in 20 uL of 0.1% TFA, sonicated 30 min or
more and spotted onto a MALDI target using 5 mg/mL
dihydroxybenzoic acid, 5 mg/mL α-cyano-4-hydroxycinnamic
acid in 50% acetonitrile 0.1% TFA as matrix.
ESI-LTQ-Orbitrap-MS
86 Da from the precursor, and then a subsequent loss of 112 Da
The crosslinked standard peptides were analyzed with a
hybrid linear quadrupole ion trap-Orbitrap mass spec-
trometer (ThermoFisher Scientific, Inc. model LTQ-Orbi-
trap XL; San Jose, CA, USA). They were introduced by
infusion using a TriVersa Nanomate nanospray ionization
source from Advion BioSciences (Ithaca, NY, USA).
Peptides were delivered with a pneumatic displacement
pressure of 0.45 psi and an applied spray voltage of
or a total 198 Da loss from the precursor. In the third filter,
potential crosslinked peptides were identified by searching the
pairs of peaks separated by 112 Da for a set in which the lower
of the two masses plus 112 Da added up to a precursor mass
observed in the spectrum. Additional MS/MS spectra were
obtained based on these results. The source code can be
obtained by contacting the authors.
1
2
.63 kV. The heated capillary temperature was set at
00 °C. Monoisotopic precursor ions were selected for
Results and Discussion
multistage tandem mass spectrometric acquisition using
Crosslinker Design
an isolation window of 3.0 (m/z units) and excitation
2
3
In the past decade, there has been an increase in research
involving chemical crosslinking combined with mass spec-
trometry, and several crosslinkers have been developed to
address one or more of the inherent difficulties associated
energy settings of 35 for both MS and 35 for MS
spectra. All CID spectra were collected in FT (Orbitrap)
mode for high mass accuracy (910 ppm) and peak
resolution (30,000 at m/z 400).
MALDI-TOF/TOF-MS
The crosslinked aldolase digest spectra were acquired in
positive reflector mode in the 4800 TOF/TOF mass
spectrometer (Applied Biosystems/MSD SCIEX). MS
spectra were acquired from 600 to 3500m/z in each
fraction. CID was achieved with a collision energy of
2
kV and atmosphere was used as the collision gas in
–
6
the medium gas setting (~10 Torr). The crosslinked
standard peptides were manually spotted onto a 4800
MALDI sample plate and then MALDI matrix spotted
over the samples (5 mg/mL of ↦-cyano-4-hydroxycin-
namic acid, 5 mg/mL 2,5-dihydroxybenzoic acid in 50%
acetonitrile, 0.1% trifluoroacetic acid). The eight most
abundant peaks in each well were automatically selected
for MS/MS fragmentation.
Scheme 1. Filtering scheme to identify crosslinked peptides

B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
205
with this technique. This project attempted to address several
of those challenges. Crosslinked peptides have been reported
to fragment poorly via CID (31, 44, 46), consistent with our
observations. For this reason and to overcome some other
limitations of current crosslinking reagents, we designed the
crosslinker to be readily cleavable by CID and in-source
decay (ISD), allowing the individual peptides released in
MS or MS (via in-source decay) to be identified via MS .
Additional desired properties included generation of distinc-
tive marker ions for the various reaction products, excellent
solubility, ease of synthesis, good reactivity, self-quenching,
and good stability in dry form. Substituted amines are
known to undergo facile fragmentation via CID and several
crosslinking and tagging reagents have been designed to
include these structures [12, 52], including commercial
reagents like the isobaric tag iTRAQ [55]. The energy required
to induce fragmentation of these structures is similar to the
energy required for peptide backbone fragmentation, allowing
cleavage to still occur along the peptide backbone. DC4
contains quaternary amine moieties to further increase cross-
linker lability over the peptide backbone and to generate
fragmentation products containing mobilized protons.
Current crosslinkers that have been used to map tertiary or
quaternary structure of proteins and complexes span a range
from the “zero length” crosslinker formaldehyde to much
longer crosslinkers of 40 or more Å [22, 24, 26]. The arm span
of DC4 was estimated to be 18Å from one leaving group to the
other, situating this crosslinker in the middle range of reported
crosslinker lengths. As discussed below for the aldolase study,
the results summarized in Table 2 clearly demonstrate that this
crosslinker is effective for lysyl residue amino groups from
approximately 18Å separation down to very short separations
as deduced from the crystal structure. For studies of very large
complexes we expect that longer crosslinkers will be needed to
complement DC4 as well as shorter crosslinks to further
constrain distances in certain cases.
enough to force the ternary reaction of two peptides with
DC4 through mass action. As expected, crosslinked pep-
tides, dead-end reaction products, and in-source decay
cleavage products from both the dead-end reaction and the
crosslinked peptide products were observed in the initial
mass spectrum via MALDI-TOF-TOF-MS. No peptides
containing unmodified lysyl residues were observed. Al-
though there are two intrinsic positive charges in the
crosslinking moiety, the mass of the crosslinked peptide
represents a loss of a proton to yield the singly charged ion
2
1
3
+
(M –H) in MALDI-TOF-MS. No evidence of a doubly
charged crosslinked peptide exists in the MALDI spectra.
This is likely due to formation of an internal ion pair
between the carboxyl group of the C-terminus and one of the
quaternary amines [56, 57]. CID of the crosslinked peptide
results in two fragments, 1050.6 and 938.5, both from
cleavage at the sites of intrinsic positive charge (Figure 2a).
The fragment at 1050.6 Da is due to cleavage at the intrinsic
positive charge on the nitrogen containing ring (DABCO),
retaining the DABCO moiety. The second fragment results
from a rearrangement reaction which eliminates the DABCO
ring. The same two cleavage products are visible in the
original mass spectrum resulting from in-source decay.
3
2
Pseudo MS (MS of the in-source decay product) of the
elimination product (938.5) produces a spectrum with a
robust series of b- and y-ions from which the component
peptide may be identified (Figure 2b). Rearrangement of
quaternary amines has been previously described by He
and Reilly [58], a similar mechanism involving a
sulfonium crosslinker was proposed by Lu et al. [47],
and the proposed mechanism for DC4 is shown here in
Figure 3. This mechanism is consistent with the data
presented here and, importantly, it mobilizes an existing
proton on the rearranged product when the original
precursor was not protonated. This mobile proton can
then facilitate backbone fragmentation yielding the b-
and y-ion series.
Tandem mass spectrometry of the dead-end product
results in a diagnostic ion clearly visible at m/z 199.1.
MS Fragmentation of Model DC4-Crosslinked
Peptides
(
Figure 4) The presence of this ion unambiguously
The N-terminally acetylated peptides Ac-QIGKGVAR and
Ac- QHAKLGR were prepared in solution concentrated
identifies this spectrum as being from a modified
peptide, although it has been observed occasionally in
Table 2. Type Two Crosslinked Peptides Identified from Proteolytic Digest of Crosslinked Aldolase. Bold Type Represents Modified Residues. The *
Represents the N-Terminus of the Protein
Crosslink type
Peptide A
Peptide B
Distance (Å)
Precursor
mass
Fragment masses
2
2
2
2
2
2
2
2
2
2
(Inter)
(Inter)
(Intra)
(Inter)
(Intra)
(Intra)
(Intra)
(Intra)
(Intra)
(Inter)
ILPDGDHDLKR (190–200)
KVLAAVYK (207–214)
YVKR (327–330)
ILPDGDHDLKR (190–200)
CVLKIGE (149–155)
YVKR (327–330)
STGSIAKR (35–42)
STGSIAKR (35–42)
*PHSHPALTPE (1–10)
YVKR (327–330)
QKKE (11–14)
QKKE (11–14)
7
10
14
15
17
23
30
30
32
39
2221.2
1920.1
1714.9
2611.3
1827.9
1631.9
1969.0
2152.1
2184.1
1507.9
651.4, 763.3, 875.4, 1278.8, 1346.8, 1458.8
959.6, 1071.7, 651.2, 763.4, 875.6
633.4, 745.5, 970.5, 1082.6
1153.6, 1265.7, 1346.7, 1458.8
887.5, 999.6, 941.5, 829.5
NLKAAQEE (319–326)
*PHSHPALTPE (1–10)
STGSIAKR (35–42)
STGSIAKR (35–42)
NLKAAQEE (319–326)
*PHSHPALTPE (1–10)
KDGADFAK (139–146)
QKKE (11–14)
999.6, 887.5, 745.5, 633.4
999.6, 887.4, 1082.5, 970.5
887.5, 999.6, 1153.5, 1265.7
1153.5, 1265.6, 1031.5
875.5, 763.4, 745.5, 633.3

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06
B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
Figure 2. (a) CID fragmentation on the MALDI-TOF-TOF of the crosslinked synthetic peptide m/z 1988.2 (homodimer of
AcQIGKGVAR) results in two major cleavage events at the intrinsic positive charges which are also observed in the first mass
3
spectrum via in-source decay. (b) Pseudo MS of the fragment without an intrinsic positive charge produces a robust series of
b- and y-ions which allows for peptide identifications
crosslinked peptides and valid crosslinked peptides may
also contain dead-end reaction products. This cannot be
used as an absolute exclusion criterion although it is
useful. The other major peaks in the MS/MS spectrum
result from fragmentation at the intrinsic positive
charges. Low level b- and y-ions are also observed,
however, the same fragments due to cleavage at the
intrinsic positive charges are present in the mass
spectrum from in-source decay. CID of the in-source
decay product which has lost the DABCO moiety yields
a robust sequence of b- and y-ions. The fragmentation of
crosslinked peptides was also evaluated in the ESI-LTQ-
Orbitrap-MS via CID and similar fragmentation events
were observed as shown in Figures 5a and b.
Algorithm Development
The in source decay fragmentation of DC4 crosslinked
peptides provides a unique series of ions that can be taken
advantage of in developing a computational approach to

B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
207
Figure 3. Although the crosslinker contains two intrinsic positive charges, peptides modified with DC4 are observed as singly
charged ions in MALDI, most likely due to the loss of a proton from a carboxylic acid moiety and ion pair formation (not shown).
Upon fragmentation of the singly charged precursor by either in-source decay or CID, the rearrangement produces two singly
charged species by mobilizing an existing proton as shown. The difference between species A and C as well as B and D is
1
12 Da resulting from the loss of the DABCO moiety and is used as a selection filter for crosslinked peptides
identifying crosslinked peptides. An algorithm was devel-
oped to identify modified peptides using a simple filtering
schema (Scheme 1). The software application based on this
algorithm initially searches MS spectra (or in-source decay
2
Figure 4. CID fragmentation via MALDI-TOF-TOF of a dead-end reaction product (m/z 1136.3) from a proteolytic digest of
crosslinked aldolase. Similar fragmentation is observed as for the crosslinked peptide (Figure 2), but a diagnostic ion at 199.1 is
observed, confirming this spectrum as being from a dead-end (type 0) product

2
08
B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
Figure 5. (a) CID on the LTQ-Orbitrap of the crosslinked homodimer synthetic peptide (Ac-QIGKGVAR) results in the same
3
two major cleavage events observed in MALDI at the intrinsic positive charges. (b) MS of the fragment without an intrinsic
positive charge produces a robust series of b- and y-ions for peptide identification
in MS spectra from MALDI) for differences of 112 Da
between two peaks. Output from this first filter may contain
dead-end peptides, crosslinked peptides, or multiply modi-
fied peptides. Next, the dead-end peptides are culled out by
searching for precursor ions that have an 86 Da loss,
followed by a 112 Da loss for a total loss of 198 Da from

B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
209
Figure 6. (a) CID of DC4-crosslinked peptides from a proteolytic digest of aldolase produces two pairs of peaks where
the lower mass is separated from the higher mass by 112 Da. The black circle represents the central DABCO moiety
and the lines on either side represent the carbon spacer arm in the crosslinker. Those same four peaks are observed in
the mass spectrum from in-source decay and CID of the lower mass of each peptide pair [(b), (c)] yields the b- and y-ions for peptide
identification
the precursors. These results are tagged as potential dead-
end peptides. The crosslinked peptides are identified by first
searching for the pairs of peaks separated by 112 Da, then the
software determines if the observed peptide pairs sum (while
correcting for the DABCO mass of 112 Da) to a precursor ion
in the spectrum. Those ions that satisfy these criteria are

2
10
B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
returned to the user as potential crosslinked peptides and are
then subjected to MS and detailed de novo analysis.
forms and portions of the molecule exhibit considerable
flexibility between crystal structures and in solution [60–63].
The other four crosslinked peptides had inter-lysyl
residue distances from 30 to 39Å. Although these distances
are longer than we would initially expect based on the length
of DC4 alone and a rigid protein structure, they are
consistent with flexibility of protein backbones and lysyl
side chains in solution. Here, several of the lysyl residues were
observed to exhibit crosslinking with more than one other lysyl
residue, consistent with earlier claims of flexibility in those
regions. This flexibility allows the crosslinker to sample a
larger region on the surface of the protein. As an example, Lys
3
Application of DC4 to Aldolase
Aldolase crosslinking by DC4 was optimized in solution by
reaction at a range of crosslinker to protein ratios and the
products evaluated by SDS gel electrophoresis (Supplemen-
tal Materials Figure 1) with the objective of maximizing
crosslinks within tetramers while minimizing crosslinks
between tetramers (nonspecific interactions). Aldolase tet-
ramers were crosslinked at the optimum ratio of DC4
41 was observed crosslinked to both Lys-152, which was 17Å
(
minimizing nonspecific interactions while maximizing
away, and Lys-321, 23Å away, but also to Lys 329, 30Å away
and to the N-terminus, also 30Å away. Deuterium exchange
studies [60] in solution indicate very high exchange rates for
the short helical region containing Lys-41 and high B-values
observed for this region in the crystal structure [61, 63] provide
further evidence for the flexibility of this region. This high level
of flexibility is consistent with the observation for Lys-41
crosslinked to Lys-321 and to the N-terminus, both at 30Å.
Likewise, the four N-terminal residues show considerable
variation between crystal structures, indicating this region is
flexible and consistent with our identification of crosslinks
between the N-terminus and Lys-199, Lys-139, and Lys-41
with distances of 15, 32, and 30Å from the N-terminus,
respectively. The crosslink exhibiting the longest span between
Lys residues is between Lys 13 and Lys 329. The latter lysyl
residue lies within the highly flexible C-terminal region
involved in binding to F-actin [62]. It has been previously
suggested that crosslinker length is not necessarily a precise
indicator of inter-residue spacing when flexibility of the
peptide backbone and lysyl side chains are factored in [64,
crosslinking efficiency) for mass spectrometry analysis.
CID of the in-source decay fragments from DC4 crosslinked
peptides generated sufficient backbone fragmentation in the
aldolase digest to identify the amino acid sequence of the
crosslinked peptides as shown in Figure 6 and in Supple-
mental Materials Figures 2–19. The majority of the spectra
from modified residues displayed preferential fragmentation,
consisting predominantly of b-ions from the N-terminal side
of the modified residues and y-ions from the C-terminal side.
Not surprisingly, this fragmentation property is somewhat
sequence dependent.
Both crosslinked peptides and dead-end peptides were
identified from proteolytic digests of DC4-crosslinked
aldolase and are shown in Tables 2 and 3, respectively.
These modified peptides were evaluated for their consisten-
cy with the crystal structure for aldolase [59], and it was
found that both the dead-end and crosslinked peptide
residues observed were solvent exposed. Six of the cross-
linked peptides identified had inter-Lys distances between 7
and 23Å. Although the DABCO moiety is rigid, the reactive
side arms are quite flexible and capable of coupling to Lys
residues, spaced closely together, consistent with the shortest
distance observed between two crosslinked Lys residues
being 7Å. The side chain residue distance measurements
were based on the rigid crystal structure and some changes in
solution phase are likely since aldolase exhibits multiple crystal
65], and our results are certainly consistent with this assertion.
All the peptides constituting type 2 crosslinked peptides
were also observed as type 0 reaction products as shown in
Table 3. Two peptides (VDKGVVPLAGTNGE, IVAPGK
GILAADE) were only observed as the type 0 products. The
closest lysyl residues for Lys-110 and Lys-27 are only 10
and 13Å away, respectively, suggesting that the crosslinked
products may not have been suitable for analysis (low
ionization efficiency or large fragment size). Two lysyl
residues, Lys-214 and Lys-146, were observed only as
unmodified forms, suggesting that they were not as accessible
to reagent as those that exhibited modification. Inspection of
these residues in the crystal structure revealed that they are
much less accessible than other lysyl residues observed to be
modified in this study, as illustrated in Supplementary Material
Figure 20, where we show a ribbon model with the pertinent
lysyl residues highlighted. The two lysyl residues are com-
pletely obscured from all angles in the space-filling model.
Table 3. The Type Zero Crosslinked Peptides (Dead-Ends) Identified from
a Proteolytic Digest of Crosslinked Aldolase. Bold Type Represents
Modified Residues. The * Represents the N-Terminus of the Protein
Peptide sequence
Precursor
mass
Fragment masses
VDKGVVPLAGTNGE (108–121)
IVAPGKGILAADE (22–34)
NLKAQEE (319–325)
KDGADFAK (139–146)
KVLAAVYK (207–214)
CVLKIGE (149–155)
1621.8
1519.8
1168.6
1117.5
1157.6
1027.5
961.5
1085.6
1351.6
831.5
1535.8, 1423.8
1433.8, 1321.7
1082.6, 970.5
1031.5, 919.5
1071.6, 959.5
941.5, 829.4
875.5, 763.4, 651.3
999.5, 887.5
1265.6, 1153.5
745.4, 633.4
QKKE (11–14)
STGIASKR (35–42)
Conclusions
We have designed and tested a new crosslinking reagent that
overcomes some of the difficulties associated with the use of
*PHSHPALTPE (1–10)
YVKR (327–330)
ILPDGDHDLKR (191–201)
1544.8
1458.7, 1346.7

B. Clifford-Nunn et. al: Cyclic Quaternary Amines as MS-Cleavable Crosslinkers
211
crosslinking reagents with mass spectrometry. The DC4
crosslinking reagent is stable as a solid over long-term
storage, highly reactive, highly soluble (1 M solutions), and
quite labile to CID. When fragmented by either CID or ISD,
DC4 crosslinked peptides fragment characteristically into
four rearranged products. Even though the precursor ions are
not protonated, the fragmentation products rearrange to
propionate), DTSSP with peptides. J. Am. Soc. Mass Spectrom. 15,
36–749 (2004)
4. Iglesias, A.H., Santos, L.F.A., Gozzo, F.C.: Collision-induced dissoci-
ation of Lys-lys intramolecular Cross-linked peptides. J. Am. Soc. Mass.
Spectrom. 20, 557–566 (2009)
5. Soderblom, E., Bobay, B., Cavanagh, J., Goshe, M.: Tandem mass
spectrometry acquisition approaches to enhance Identification of
Protein–protein interactions using Low-energy Collision-induced disso-
ciative chemical crosslinking reagents. Rapid Commun. Mass Spectrom.
21, 3395–3408 (2007)
6. Kalkhof, S., Sinz, A.: Chances and pitfalls of chemical Cross-linking
with Amine-reactive N-hydroxysuccinimide Esters. Anal. Bioanal.
Chem. 392, 305–312 (2008)
7. Madler, S., Bich, C., Touboul, D., Zenobi, R.: Chemical Cross-linking
with NHS Esters: A systematic study on Amino acid reactivities. J.
Mass Spectrom. 44, 694–706 (2009)
18. Zhang, Q., Crosland, E., Fabris, D.: Nested Arg-specific bifunctional
crosslinkers for MS-based structural analysis of proteins and protein
assemblies. Anal. Chim. Acta 627, 117–128 (2008)
7
1
1
3
generate a mobile proton, and MS results in efficient
1
1
backbone fragmentation for peptide identification. Database
searches can then identify which peptides were crosslinked,
and based on this characteristic fragmentation behavior, an
algorithm was developed to identify which peptides were
crosslinked. This crosslinking strategy was used on the
model tetrameric protein, aldolase, and the crosslinked
peptides identified were consistent with the structure.
1
9. Novak, P., Kruppa, G.H.: Intra-molecular Cross-linking of acidic
residues for protein structure studies. Eur. J. Mass Spectrom. 14, 355–
3
65 (2008)
2
0. Krauth, F., Ihling, C.H., Ruttinger, H.H., Sinz, A.: Heterobifunctional
Isotope-labeled Amine-reactive Photo-cross-linker for structural inves-
tigation of proteins by MALDI-TOF/TOF and ESI-LTQ-orbitrap mass
spectrometry. Rapid Commun. Mass Spectrom. 23, 2811–2818 (2009)
Acknowledgments
The authors acknowledge funding in part for this work by
NIH #1P41RR018627 and NIH #R01GM09583201. They
thank the members of the Michigan Proteome Consortium
for their assistance in this effort. Peptides were synthesized
by the Biomedical Research Core Facility. The authors
acknowledge that Bryan E. Smith wrote the filtering
algorithm for identifying candidate crosslinked peptides.
21. Kang, S., Mou, L., Lanman, J., Velu, S., Brouillette, W.J., Prevelige Jr.,
P.E.: Synthesis of Biotin-tagged chemical Cross-linkers and their
applications for mass spectrometry. Rapid Commun. Mass Spectrom.
2
3, 1719–1726 (2009)
22. Zhang, H., Tang, X., Munske, G.R., Tolic, N., Anderson, G.A., Bruce,
J.E.: Identification of Protein–protein interactions and topologies in
living cells with chemical Cross-linking and mass spectrometry. Mol.
Cell. Proteom. 8, 409–420 (2009)
2
3. Fujii, N., Jacobsen, R.B., Wood, N.L., Schoeniger, J.S., Guy, R.K.: A
novel protein crosslinking reagent for the determination of moderate
resolution protein structures by mass spectrometry (MS3D). Bioorg
Med. Chem. Lett. 14, 427–429 (2004)
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