Chromogenic cross-linker for the characterization of protein structure by infrared multiphoton dissociation mass spectrometry

Article abstract of DOI:10.1021/ac800625x

We have developed a new IR chromogenic cross-linker (IRCX) to aid in rapidly distinguishing cross-linked peptides from unmodified species in complex mixtures. By incorporating a phosphate functional group into the cross-linker, one can take advantage of i

Full text of DOI:10.1021/ac800625x

ac research
Anal. Chem. 2008, 80, 4807–4819
Accelerated Articles
Chromogenic Cross-Linker for the Characterization
of Protein Structure by Infrared Multiphoton
Dissociation Mass Spectrometry
Myles W. Gardner, Lisa A. Vasicek, Shagufta Shabbir, Eric V. Anslyn, and Jennifer S. Brodbelt*
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300,
Austin, Texas, USA 78712
We have developed a new IR chromogenic cross-linker
(IRCX) to aid in rapidly distinguishing cross-linked pep-
tides from unmodified species in complex mixtures. By
incorporating a phosphate functional group into the cross-
linker, one can take advantage of its unique IR absorption
properties, affording selective infrared multiphoton dis-
sociation (IRMPD) of the cross-linked peptides. In a mock
mixture of unmodified peptides and IRCX-cross-linked
peptides (intramolecularly and intermolecularly cross-
linked), only the peptides containing the IRCX modifica-
tion were shown to dissociate upon exposure to 50 ms of
10.6-µm radiation. LC-IRMPD-MS proved to be an effec-
tive method to distinguish the cross-linked peptides in a
tryptic digest of IRCX-cross-linked ubiquitin. A total of
four intermolecular cross-links and two dead-end modi-
fications were identified using IRCX and LC-IRMPD-MS.
IRMPD of these cross-linked peptides resulted in second-
ary dissociation of all primary fragment ions containing
the chromophore, producing a series of unmodified b- or
y-type ions that allowed the cross-linked peptides to be
sequencedwithouttheneedforcollision-induceddissociation.
protein-protein interactions.3–6 Compared to X-ray crystallography
and NMR spectroscopy, chemical cross-linking requires relatively
low amounts of protein to probe distance constraints within a
single protein7 or protein-ligand complexes.8–10 While the
experimental procedure for chemically cross-linking proteins is
well established, there are several analytical challenges that
remain in the identification of the cross-linked peptides by mass
spectrometric means. The two main difficulties in this technique
have been the differentiation of cross-linked peptides from
unmodified ones in enzymatic digests of cross-linked proteins and
the ability to identify the specific location of the cross-link using
tandem MS.
To circumvent these limitations, much work has been dedi-
cated to developing new cross-linkers to simplify identification by
mass spectrometric methods. For example, several cross-linkers
that incorporate isotope labels have been designed to create
doublet peaks within the mass spectra,11–14 thus allowing facile
pinpointing of the cross-linked products. Other cross-linkers
(3) Back, J. W.; De Jong, L.; Muijsers, A. O.; De Koster, C. G. J. Mol. Biol.
2003, 331, 303–313
(4) Sinz, A. Mass Spectrom. Rev. 2006, 25, 663–682
(5) Sinz, A. J. Mass Spectrom. 2003, 38, 1225–1237
.
.
.
(6) Young, M. M.; Tang, N.; Hempel, J. C.; Oshiro, C. M.; Taylor, E. W.; Kuntz,
I. D.; Gibson, B. W.; Dollinger, G. Proc. Natl. Acad. Sci. U. S. A. 2000, 97,
As the field of proteomics continues to rapidly expand, there
has been a growing interest in determining the three-dimensional
structure of proteins as well as the interfaces of protein-protein
complexes.1,2 Chemical cross-linking of proteins with mass
spectrometric analysis has proven to be a useful method to
determine low-resolution protein structural information and
5802–5806
(7) Huang, B. X.; Kim, H.-Y.; Dass, C. J. Am. Soc. Mass Spectrom. 2004, 15,
1237–1247
(8) Carlsohn, E.; Angstrom, J.; Emmett, M. R.; Marshall, A. G.; Nilsson, C. L.
Int. J. Mass Spectrom. 2004, 234, 137–144
(9) Lanman, J.; Lam, T. T.; Barnes, S.; Sakalian, M.; Emmett, M. R.; Marshall,
A. G.; Prevelige, P. E. J. Mol. Biol. 2003, 325, 759–772
.
.
.
.
(10) Back, J. W.; Sanz, M. A.; De Jong, L.; De Koning, L. J.; Nijtmans, L. G. J.;
* To
jbrodbelt@mail.utexas.edu.
(1) Borch, J.; Jorgensen, T. J. D.; Roepstorff, P. Curr. Opin. Chem. Biol. 2005,
9, 509–516
(2) Heck, A. J. R.; van den Heuvel, R. H. H. Mass Spectrom. Rev. 2004, 23,
368–389
whom
correspondence
should
be
addressed.
E-mail:
De Koster, C. G.; Grivell, L. A.; Van Der Spek, H.; Muijsers, A. O. Protein
Sci. 2002, 11, 2471–2478
(11) Mueller, D. R. ; Schindler, P. ; Towbin, H. ; Wirth, U. ; Voshol, H. ; Hoving,
S. ; Steinmetz, M. O. Anal. Chem. 2001, 73, 1927–1934
(12) Back, J. W.; Notenboom, V.; de Koning, L. J.; Muijsers, A. O.; Sixma, T. K.;
de Koster, C. G.; de Jong, L. Anal. Chem. 2002, 74, 4417–4422
.
.
.
.
.
10.1021/ac800625x CCC: $40.75 2008 American Chemical Society
Published on Web 06/03/2008
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 4807

incorporate fluorophores to facilitate distinction of modified
proteins15 and peptides from the unmodified ones during LC-MS
analyses,16,17 while others introduce chemically cleavable func-
tionalities.18–20 Affinity tags such as biotin labels have also shown
promise as a means to enrich the cross-linked peptides from
complex mixtures prior to mass spectrometry.20–22 Cross-linkers
have also been designed with gas-phase labile bonds such that
traceable fragment ions unique to cross-linked peptides are formed
upon low-energy activation during MS/MS analysis.20,23–25
Interpretation of the cross-linked peptide product ion spectra,
which typically contain diagnostic fragment ions both to locate
the cross-link and to sequence the peptides, remains a difficult
challenge even with available software. Several algorithms have
been written to aid in the identification of intact cross-linked
peptides based on product ion spectra of these species, but they
do not take into account all of the possible product ions specific
to cross-linked peptides.26–31 Initial work by Schilling et al.
demonstrated that dissociation of cross-linked peptides typically
resulted in cleavage of an amide bond of one peptide, yielding
one unmodified fragment and the corresponding fragment linked
to the other peptide.26 A recent systematic study in our laboratory
indicated that intermolecularly cross-linked peptides without a
mobile proton yielded a high degree of internal ions, as well as
other double-cleavage products, which make interpretation of the
product ion spectra difficult.32 Thus, developing new chemical
cross-linkers to reduce the complexity of the product ion spectra
is still an ongoing area of interest. Recent work by Soderblom et
al. reported the design of a cross-linker in which a gas-phase labile
bond was inserted in the chemical cross-linker, thus cleaving upon
low-energy collisions to yield the two constituent peptides.33,34
Bruce and co-workers developed a similar strategy using cross-
linkers with two labile bonds that upon dissociation produce the
two peptides in addition to a diagnostic reporter fragment ion.24,25
After cleavage of the labile bond of these cross-linkers, the two
modified peptides are then further interrogated via MS3 to
sequence each peptide.
Our aim is to develop a chemical cross-linking technique in
conjunction with photodissociation methods to eliminate the need
for MSn
experiments and streamline the analysis of cross-linked
peptides. Infrared multiphoton dissociation (IRMPD) has shown
promise as an alternative means to activate and dissociate
biomolecules in the gas phase35,36 and has been shown to be an
effective means to selectively dissociate phosphopeptides in both
FTICR37,38 and quadrupole ion trap (QIT) mass spectrometers.39,40
Previous work in our laboratory39 and by Flora and Muddiman37,38,41
have shown that the phosphate group has a high absorption at
the wavelength of a continuous wave CO2 laser, 10.6 µm. Because
of the large differences in absorption efficiency of phosphopeptides
and unmodified peptides, phosphopeptides dissociate upon IR
irradiation far more readily than unmodified peptides.
IRMPD in QITs offers other advantages over conventional
collisional activation methods.36,42–47 IRMPD is a nonresonant
activation process whose efficiency is independent of the rf
trapping voltage, thus allowing a much broader m/z trapping range
than conventional CID. This feature is particularly important for
detection of key terminal sequence ions in the low m/z range.
Ion losses due to collisions or unstable trajectories are eliminated
as the photodissociation process does not affect the translational
motion of ions. The nonresonant nature of photodissociation
methods also yields secondary and higher order product ions that
might only be obtained by MSn approaches using CID.
In the present study, we have developed a novel IR-chromoge-
nic cross-linker that incorporates a phosphate chromophore that
allows the identification of cross-linked peptides and facilitates the
interpretation of the product ion spectra of the constituent
peptides. IRMPD in a linear quadrupole ion trap is used to
selectively dissociate the cross-linked peptides, allowing for these
peptides to be easily distinguished from unmodified peptides (i.e.,
ones that do not afford any protein contact information). The
resulting IRMPD spectra of intermolecularly cross-linked peptides
(13) Chu, F.; Mahrus, S.; Craik, C. S.; Burlingame, A. L. J. Am. Chem. Soc. 2006,
128, 10362–10363
.
(14) Ihling, C.; Schmidt, A.; Kalkhof, S.; Schulz, D. M.; Stingl, C.; Mechtler, K.;
Haack, M.; Beck-Sickinger, A. G.; Cooper, D. M. F.; Sinz, A. J. Am. Soc.
Mass Spectrom. 2006, 17, 1100–1113.
(15) Wine, R. N. ; Dial, J. M. ; Tomer, K. B. ; Borchers, C. H. Anal. Chem. 2002,
74, 1939–1945
(16) Sinz, A.; Wang, K. Biochemistry 2001, 40, 7903–7913
(17) Sinz, A.; Wang, K. Anal. Biochem. 2004, 331, 27–32
(18) Bennett, K. L.; Kussmann, M.; Bjork, P.; Godzwon, M.; Mikkelsen, M.;
Sorensen, P.; Roepstorff, P. Protein Sci. 2000, 9, 1503–1518
(19) Petrotchenko, E. V.; Olkhovik, V. K.; Borchers, C. H. Mol. Cell. Proteomics
2005, 4, 1167–1179
(20) Trester-Zedlitz, M.; Kamada, K.; Burley, S. K.; Fenyoe, D.; Chait, B. T.;
Muir, T. W. J. Am. Chem. Soc. 2003, 125, 2416–2425
(21) Hurst, G. B.; Lankford, T. K.; Kennel, S. J. J. Am. Soc. Mass Spectrom. 2004,
15, 832–839
(22) Sinz, A.; Kalkhof, S.; Ihling, C. J. Am. Soc. Mass Spectrom. 2005, 16, 1921–
1931
(23) Back, J. W.; Hartog, A. F.; Dekker, H. L.; Muijsers, A. O.; de Koning, L. J.;
de Jong, L. J. Am. Soc. Mass Spectrom. 2001, 12, 222–227
(24) Tang, X.; Munske, G. R.; Siems, W. F.; Bruce, J. E. Anal. Chem. 2005, 77,
311–318
(25) Chowdhury, S. M.; Munske, G. R.; Tang, X.; Bruce, J. E. Anal. Chem. 2006,
78, 8183–8193
(26) Schilling, B.; Row, R. H.; Gibson, B. W.; Guo, X.; Young, M. M. J. Am. Soc.
Mass Spectrom. 2003, 14, 834–850
(27) Gao, Q.; Xue, S.; Doneanu, C. E.; Shaffer, S. A.; Goodlett, D. R.; Nelson,
S. D. Anal. Chem. 2006, 78, 2145–2149
(28) Chen, T.; Jaffe, J. D.; Church, G. M. J. Comput. Biol. 2001, 8, 571–583
(29) Tang, Y.; Chen, Y.; Lichti, C. F.; Hall, R. A.; Raney, K. D.; Jennings, S. F.
BMC Bioinformatics 2005, 6, S9
.
.
.
.
.
.
.
(33) Soderblom, E. J.; Bobay, B. G.; Cavanagh, J.; Goshe, M. B. Rapid Commun.
.
Mass Spectrom. 2007, 21, 3395–3408
(34) Soderblom, E. J.; Goshe, M. B. Anal. Chem. 2006, 78, 8059–8068
(35) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W.
Anal. Chem. 1994, 66, 2809–2815
(36) Payne, A. H.; Glish, G. L. Anal. Chem. 2001, 73, 3542–3548
(37) Flora, J. W.; Muddiman, D. C. Anal. Chem. 2001, 73, 3305–3311
(38) Flora, J. W.; Muddiman, D. C. J. Am. Chem. Soc. 2002, 124, 6546–6547
(39) Crowe, M. C.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2004, 15, 1581–
1592
(40) Crowe, M. C.; Brodbelt, J. S. Anal. Chem. 2005, 77, 5726–5734
(41) Flora, J. W.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2004, 15, 121–
127
(42) Colorado, A.; Shen, J. X.; Brodbelt, J. Anal. Chem. 1996, 68, 4033–4043
(43) Gabryelski, W.; Li, L. Rapid Commun. Mass Spectrom. 2002, 16, 1805–
1811
(44) Goolsby, B. J.; Brodbelt, J. S. Anal. Chem. 2001, 73, 1270–1276
(45) Hashimoto, Y.; Hasegawa, H.; Waki, I. Rapid Commun. Mass Spectrom.
2004, 18, 2255–2259
(46) Stephenson, L., Jr.; Booth, M. M.; Shalosky, J. A.; Eyler, J. R.; Yost, R. A.
J. Am. Soc. Mass Spectrom. 1994, 5, 886–893
(47) Wilson, J. J.; Brodbelt, J. S. Anal. Chem. 2006, 78, 6855–6862
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
(30) de Koning, L. J.; Kasper, P. T.; Back, J. W.; Nessen, M. A.; Vanrobaeys, F.;
.
Van Beeumen, J.; Gherardi, E.; de Koster, C. G.; de Jong, L. FEBS J. 2006,
.
273, 281–291
(31) Seebacher, J.; Mallick, P.; Zhang, N.; Eddes, J. S.; Aebersold, R.; Gelb, M. H.
J. Proteome Res. 2006, 5, 2270–2282
(32) Gardner, M. W.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2008, 19, 344–
357
.
.
.
.
.
.
4808 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

ature for 24 h and then extracted with H2O (1 × 60 mL), saturated
NaHCO3 (1 × 60 mL), and then a saturated brine solution (1 × 60
mL). The final liquid extract was dried over Na2SO4, filtered, and
dried under vacuum. The intermediate was recrystallized in DCM
at 20 °C overnight and filtered.
Dibenzoyloxysuccinimidyl ethyl phosphate was prepared by
dissolving 0.5 mmol of N-(4-hydroxybenzoyloxy)succinimide in
dry THF at 0 °C and then adding 0.25 mmol of ethyl dichloro-
phosphate dropwise to the solution, followed by 10 mmol of dry
triethylamine. The reaction was allowed to warm to room tem-
perature while stirring over 2 h, and then was heated to 60 °C
and stirred for additional 4 h. The final solution was dried and
concentrated under reduced pressure. The product was washed
with cold H2O, filtered, recrystallized in DCM, and filtered. The
cross-linker structure was confirmed by 1H NMR using a 400-
1
MHz Varian instrument. H NMR at 400 MHz in CDCl3 yielded
the following: 8.29 (d, 4H), 7.39 (d, 4H), 4.20 (m, 2H), 2.94 (s,
8H), 1.71 ppm (br, 3H); these findings are consistent with the
proposed structure. ESI-IRMPD-MS of protonated IRCX (m/z 561)
using 2.0 ms of irradiation yielded an abundant product ion
corresponding to the loss of succinimide (m/z 464) and a less
abundant ion of m/z 446 (loss of N-hydroxysuccinimide) (data
not shown).
Chemical Cross-Linking of Model Peptides. Stock solutions
of the model peptides were prepared at 5.0 mM in H2O. IRCX
and DSS were freshly prepared at 20.0 mM in DMSO prior to
cross-linking. The peptides R-MSH and Ac-RFMWMK-NH2 were
intermolecularly cross-linked with IRCX at a 4:4:5 molar ratio of
peptide/peptide/cross-linker in 20 mM HEPES, pH 8.0, to produce
a final concentration of 1.7 mM in a total volume of 12 µL.
Substance P was similarly intramolecularly cross-linked by IRCX
or DSS at a 4:5 molar ratio in the same buffer. All peptide cross-
linking reactions were allowed to incubate at room temperature
overnight to increase the yield of cross-linked peptides. The
reactions were desalted by solid-phase extraction using 50 mg of
tC18 Waters (Milford, MA) Sep-Pak cartridges.
Figure 1. Structures of IRCX (A), an IRCX cross-link (B), and an
IRCX-OH modification (C).
are dominated by b- and y-type product ions of each constituent
peptide. Online liquid chromatography (LC)-IRMPD-MS has been
applied for the screening of complex peptide mixtures, including
tryptic digests of chemically cross-linked proteins.
EXPERIMENTAL SECTION
Chemicals and Reagents. The peptides R-MSH (Ac-SYSME-
HFRWGKPV-NH2), Ac-RFMWMK-NH2, substance P (RPKPQQFF-
GLM-NH2), and neurotensin 1-6 (Pyr-LYENK) were purchased
from Bachem (Torrance, CA). The peptide angiotensin II (DRVYI-
HPF) was purchased from AnaSpec (San Jose, CA). Ubiquitin from
bovine red blood cells, proteomics grade trypsin, bradykinin
(RPPGFSPFR), methionine-enkephalin (YGGFM), melittin, and
the cross-linker disuccinimidyl suberate (DSS) were obtained from
Sigma (St. Louis, MO). N-Hydroxysuccinimide, 4-hydroxybenzoic
acid, and ethyl dichlorophosphate were from Aldrich (Milwaukee,
WI). Water (HPLC grade) and acetonitrile (HPLC grade) were
from Riedel-de Hae¨n (Seelze, Germany). 1-Ethyl-3-(3-dimethy-
laminopropyl)carbodiimide hydrochloride was obtained from
Pierce Biotechnology (Rockford, IL). All other chemicals and
solvents were purchased from Fisher Scientific (Fairlawn, NJ)
except for formic acid (EM Science, Gibbstown, NJ).
Synthesis of Dibenzoyloxysuccinimidyl Ethyl Phosphate.
The IR chromogenic cross-linker (IRCX, Figure 1A), dibenzoy-
loxysuccinimidyl ethyl phosphate, was synthesized by first prepar-
ing the intermediate N-(4-hydroxybenzoyloxy)succinimide. Under
an argon atmosphere, dry tetrahydrofuran (THF) (45 mL) and
dry dichloromethane (DCM) (45 mL) were added to 5 mmol of
4-hydroxybenzoic acid, 8 mmol of N-hydroxysuccinimide, and 6.5
mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride. The reaction was stirred under argon at room temper-
Chemical Cross-Linking and Enzymatic Digestion of
Ubiquitin. The protein ubiquitin was intramolecularly cross-linked
by IRCX at 1:5, 1:10, 1:25, and 1:50 molar ratios of protein/cross-
linker in 20 mM HEPES buffer, pH 8.0, at a final protein
concentration of 10 µM. The reactions were allowed to proceed
at room temperature for 30 min. The cross-linking was quenched
by the addition of NH4HCO3 to a final concentration of 10 mM
for 15 min. The reaction mixtures were desalted against a 5-kDa
molecular mass cutoff Ultrafree Biomax membrane (Millipore,
Billerica, MA). Aliquots were removed for direct-ESI-MS analysis,
and the remaining cross-linked ubiquitin sample was diluted to
180 µM in 100 mM NH4HCO3, pH 8.0. Cross-linked ubiquitin was
enzymatically digested with trypsin at a 1:10 ratio of enzyme to
protein (w/w) overnight at 37 °C. The digested sample was then
desalted by solid-phase extraction as described above and diluted
to ∼10 µM in 50:50 H2O/MeOH.
Mass Spectrometry and Infrared Multiphoton Dissocia-
tion. All mass spectrometry experiments were performed on a
modified ThermoFisher LTQ XL linear ion trap mass spec-
trometer using the XCalibur version 2.2 software package and
the standard ESI source. For direct infusion experiments, the
ESI voltage was set to 4.5 kV and solutions were infused at 2.5
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 4809

µL/min. Solutions were prepared at ∼10 µM in 49.5:49.5:1 H2O/
MeOH/HOAc (v/v/v) for ESI-MS analysis. For CID experi-
ments, the precursor ions were activated for 30 ms at the default
q-value of 0.25. Energy-variable CID experiments were per-
formed by incrementally raising the CID voltage, and the CID
voltage necessary to reduce the precursor ion abundance to
50% was determined. For energy-variable CID, normalized
collision energies were converted to CID voltages to account
for the differences in m/z of the precursor ions based on the
calibration settings of the instrument. The CID voltages for
the IRCX-cross-linked peptides were then corrected for their
greater degrees of freedom compared to the DSS-cross-linked
analogues using the following formula:
this range were isolated for 50 ms but not subjected to any form
of activation, and then an IRMPD spectrum of all ions of m/z
450-2000 with an irradiation time of 50 ms. For the isolation and
IRMPD spectra, the q-value was set to 0.18 for ions of m/z 450,
thus establishing the lower m/z limit as 90. The second LC-
IRMPD-MS experiment employed data-dependent MS/MS in
which IRMPD and CID spectra were acquired for the two most
abundant ions observed in the full mass spectum. IRMPD was
performed for 30 ms with the q-value of the precursor ion set to
0.1 (lower m/z limit ranged from 50 to 175 depending on the
precursor ion), and CID was performed for 30 ms at a normalized
collision energy of 45% using a precursor ion q-value of 0.25 (lower
m/z limit 115 - 455).
Identification of Cross-Linked Peptides. Product ion spec-
tra of peptide ions exhibiting high IRMPD efficiency were
manually identified aided in part by the software program
ProteinXXX, the protein cross-linking function of GPMAW (Gen-
eral Protein Mass Analysis for Windows) version 7.10 (Lighthouse
Data, Odense, Denmark) (available at http://www.gpmaw.com).
This program was used to provide a list of intact masses of
possible cross-linked tryptic peptides allowing for cleavage C-
terminal to only arginine and unmodified lysine residues (i.e., no
cleavage C-terminal to cross-linked or dead-end modified lysine
residues). The intact mass of the eluting species was matched to
the list potential candidates and then the identity of the precursor
ion was confirmed manually by IRMPD; all product ions were also
identified manually.
NDSS
NIRCX
V50%,corrected ) V50%
where the degrees of freedom N ) 3n - 6 and n is the number
of atoms in the peptide cross-linked by either DSS or IRCX.48 All
energy-variable CID experiments were performed in triplicate; the
drifts in the CID voltages were determined to be less than 0.08
mV.
Infrared Multiphoton Dissociation. IRMPD was performed
using a model 48-5 Synrad 50-W CO2 continuous wave laser
(Mukilteo, WA). The back flange of the vacuum manifold of the
instrument was modified with a CF viewport flange with a ZnSe
window to allow the transmission of 10.6-µm radiation. The
unfocused laser beam was aligned on axis with the linear ion trap
such that the beam passed through the 2-mm aperture of the exit
lens to irradiate the ion cloud. The laser was triggered during
the activation step in the scan function by a TTL signal from pin
14 of the J1 connector on the digital printed circuit board. All
IRMPD experiments were performed at full power (50 W) with
irradiation times varying between 5 and 100 ms unless otherwise
indicated. The q-value of the precursor was set between 0.1 and
0.15 to reduce the low-mass cutoff value to less than m/z 150.
The pressure in the analyzer region was nominally 9.0 × 10-6 Torr,
and no changes to the He bath gas pressure were made.
Analytical High-Performance Liquid Chromatography and
Tandem Mass Spectrometry. . Liquid chromatography was
performed using a Hitachi L-7000 (Hitachi Ltd.) system including
an L-7100 HPLC pump and L-7000 autosampler; the LC system
was controlled using the Hitachi 3DQ software. Reversed-phase
HPLC was accomplished using a Symmetry300 (Waters, Milford,
MA) C18 column (2.1 × 50 mm, 3.5-µm packing) with a Sym-
metry300 C18 guard column (2.1 × 10 mm, 3.5 µm packing). For
analytical separations, a gradient elution of mobile phases consist-
ing of (A) H2O with 0.1% formic acid and (B) acetonitrile with
0.1% formic acid was used at a flow rate of 0.300 mL/min. After
loading the sample (10-25-µL injections of 10 µM solutions), the
mobile phase was held at 95% A for 2 min and then the peptides
were eluted using a linear gradient from 95 to 50% A over 58 min.
Two types of online LC-IRMPD-MS experiments were per-
formed. The first data-acquisition sequence employed the follow-
ing collection: a full mass spectrum of m/z 300-2000, then an
isolation spectrum of m/z 450-2000 in which all ions of m/z in
RESULTS AND DISCUSSION
Cross-Linker Design and Characterization. It has previ-
ously been demonstrated that phosphorylated peptides can be
selectively dissociated via IRMPD in a quadrupole ion trap.39,40
Our cross-linker (Figure 1A) incorporated a phosphate chro-
mophore due to its greater IR absorption efficiency at 10.6
µm.37–39,41 The chromophore was placed in the center of the cross-
linker, thus creating a phosphate triester to avoid the neutral loss
of phosphoric acid, which is commonly observed upon ion
activation of phosphorylated peptides.49–52 The cross-linker was
designed as a homobifunctional reagent with two terminal NHS-
esters that react with primary amines (e.g., ꢀ-amine of lysine
residues or free N-terminus) similar to most common commercial
cross-linkers. IRCX has a spanner arm length of ∼16 Å and could
span a distance up to ∼28 Å between R-carbons of two lysine
residues in proteins. Upon reaction of each NHS-ester with a
primary amine, stable amide bonds are formed, yielding a cross-
link corresponding to a mass addition of 330 Da (Figure 1B). If
one NHS-ester does not react with the protein or peptide and is
hydrolyzed, a dead-end modification is produced, termed IRCX-
OH as shown in Figure 1C, with a mass addition of 348 Da.
Product ions formed upon dissociation of the cross-linked peptides
were labeled according to previously suggested nomen-
(49) Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. J. Am. Soc. Mass
Spectrom. 1993, 4, 710–717
(50) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239,
180–192
(51) DeGnore, J. P.; Qin, J. J. Am. Soc. Mass Spectrom. 1998, 9, 1175–1188
(52) Szczepanowska, J.; Zhang, X.; Herring, C. J.; Qin, J.; Korn, E. D.; Brzeska,
H. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 8503–8508
.
.
.
(48) Jones, J. L.; Dongre, A. R.; Somogyi, A.; Wysocki, V. H. J. Am. Chem. Soc.
1994, 116, 8368–8369
.
.
4810 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

clature.26,53 Briefly, for intermolecularly cross-linked peptides, the
two constituent peptides are labeled R and ꢀ, referring to the first
and second peptides, respectively. Cleavage along the backbone
of the R-peptide yields products ions such as ynR or bnR, and similar
nomenclature is used for the ꢀ-peptide and other product ion
types. For intramolecularly cross-linked peptides, the nomencla-
ture is identical to that of unmodified peptides. In addition, lysine
immonium ions that are linked to the other peptide are labeled
KLR, where L represents the cross-linker and R is the other
peptide. Similar nomenclature is used for referring to internal ions
of one peptide linked to the second peptide, or a fragment ion of
without the modification (e.g., bnR+ and bnꢀ+ ions). Fragment ions
that contain the IRCX cross-link still retain the phosphate chro-
mophore and thus can undergo IR photon absorption and
secondary dissociation. CID predominantly yielded product ions
2+
that retained the cross-link (e.g., series of ynR ions), whereas
these fragment ions were less abundant in the IRMPD spectrum.
Time-resolved IRMPD was performed in order to investigate the
relative abundances of the product ion types observed by varying
the irradiation time from 0 to 100 ms. For each IRMPD spectrum
acquired, the peak areas of the various product ion typessthose
containing or not containing the IRCX cross-linkswere measured
and summed as a function of irradiation time. The time-resolved
IRMPD plot in Figure 4 indicates that for irradiation times less
than 10 ms the most abundant product ions observed were IRCX-
cross-linked fragments. With longer irradiation times, these
product ions decreased in relative abundance, forming unmodified
b- and y-type ions, and after ∼30 ms of irradiation, virtually no
IRCX-containing product ions survived. Using IRMPD, one can
thus circumvent many dead-end dissociation pathways such as
dehydration because the primary IRCX-containing fragment ions
undergo secondary dissociation to produce more informative
fragment ions.
Upon reaction with IRCX, the peptide substance P was
intramolecularly cross-linked between the free N-terminus and
Lys-3. The doubly protonated IRCX-cross-linked substance P
underwent efficient photodissociation using 50 ms of irradiation
at only 35 W (Figure 5A). This result, in conjunction with those
described above, indicates that both intramolecularly and inter-
molecularly IRCX-cross-linked peptides can be readily photodis-
sociated. A series of singly charged b-ions were observed along
with the dominant y8 ion, a result of cleavage C-terminal to the
intramolecular cross-link, which has previously been noted as a
site of enhanced dissociation.53 Collisionally induced dissociation
of [substance P X IRCX + 2H]2+, as shown in Figure 5B, yielded
sequence coverage similar to that of IRMPD, suggesting that
either method is suitable for identifying the site of the cross-link.
However, in the CID spectrum, the b-ions retaining the IRCX
cross-link were observed in both the 1+ and 2+ charge state (e.g.,
b82+, b92+, and b102+), cluttering the product ion spectrum, and
fragment ions of low m/z had much lower relative abundances
than in the IRMPD spectrum. Upon IR irradiation, the more highly
charged IRCX-containing b-ions apparently undergo secondary
dissociation at a faster rate than the singly charged ions due to
the presence of a mobile proton. In addition, the expanded m/z
storage range possible during IRMPD allowed the detection of
the low-mass y1 ion, which was not observed by CID. For
precursor ions of higher m/z, the low-mass cutoff inherent to CID
would be alleviated by utilizing IRMPD, thus allowing other
product ions to be observed that would otherwise be undetectable
by CID.
L
the second peptide (e.g., b4R y7ꢀ).
IRMPD Analysis of IRCX Cross-Linked Peptides. The
peptides R-MSH (R-peptide) and Ac-RFMWMK-NH2 (ꢀ-peptide)
were incubated with IRCX, yielding all three possible intermo-
lecularly cross-linked products (R X R, ꢀ X ꢀ, R X ꢀ). The two
constituent peptides each have only a single reactive site for the
cross-linking reaction, Lys-11 of R-MSH and Lys-6 of Ac-RFM-
WMK-NH2 as the N-termini of each of these peptides are
acetylated, allowing for the site of the cross-link to be known with
certainty for these initial studies. It was possible to readily screen
for the cross-linked peptides in the product mixture by acquiring
an isolation spectrum in which ions of m/z 400-2000 were isolated
for 30 ms without activation by toggling off the isolation waveform,
and subsequently acquiring an IRMPD spectrum of ions in the
same m/z range by triggering the CO2 laser for those 30 ms
(Figure 2). Upon IR irradiation, only the peptide ions containing
an IRCX cross-link or IRCX-OH modification decreased in ion
abundance, allowing rapid screening of the IRCX-containing ions
of interest. All of the cross-linked peptide ions, regardless of
charge state, reduced in ion abundance by more than 95%, as well
as the peptide ions with a dead-end IRCX-OH modification. The
unmodified peptides did not undergo photodissociation, and no
decrease in their ion abundances was observed. After pinpointing
the IRCX-cross-linked peptide ions, they can be more closely
interrogated by IRMPD, CID, or both. For example, the intermo-
lecularly cross-linked peptide [R-MSH X IRCX X Ac-RFMWMK-
NH2 + 3H]3+ was analyzed by IRMPD to determine the location
of the cross-link. As shown in Figure 3A, the IRCX-cross-linked
peptide underwent very efficient dissociation upon 12 ms of
irradiation, and broad sequence coverage of each constituent
peptide was observed. CID of the same ion produced similar
sequence coverage of each peptide; however, in contrast the triply
and doubly charged b11R product ions of m/z 907.5 and 1360.4
dominated the spectrum. Previous work in our laboratory has
shown that a proline residue positioned N-terminal to the cross-
linked lysine enhances dissociation of the amide bond between
the lysine and proline, in this case producing the b11R fragment.32
Most of the product ions observed in the CID spectrum had
relative abundances of less than 1%, making it difficult to
distinguish these peaks from noise. In addition, due to the limited
m/z storage range during CID, the diagnostic y2R product ion of
m/z 214.0 cannot be trapped and detected.
The CID voltages determined from energy-variable CID
experiments performed on both DSS- and IRCX-cross-linked
peptides indicate the IRCX-cross-linked species have higher
dissociation energies than the DSS-cross-linked analogues. For
example, for the intermolecularly cross-linked R-MSH and Ac-
RFMWMK-NH2 in the 3+ charge state, the IRCX species had a
higher CID voltage than the DSS analogue by 3.7 mV (32.8 versus
29.1 mV). Likewise, the doubly protonated IRCX-intramolecularly
Upon closer examination of the IRMPD spectrum in Figure
3A, there is a distinct difference between the relative abundances
of the product ions containing the IRCX cross-link and those
(53) Gaucher, S. P.; Hadi, M. Z.; Young, M. M. J. Am. Soc. Mass Spectrom. 2006,
17, 395–405
.
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 4811

Figure 2. Isolation (A, C) and IRMPD (B, D) spectra of IRCX cross-linking product mixture of R-MSH (R-peptide) and Ac-RFMWMK-NH2
(ꢀ-peptide). IRCX cross-links are represented by X and IRCX-OH dead-end modifications by ꢁ. Ions were either isolated with the laser off (A,
C) or irradiated for 30 ms at 50 W (B, D). Zoomed in region of m/z 700-1040 shown in (C) and (D).
cross-linked substance P required a CID voltage of 1.8 mV greater
than that of the analogous DSS-peptide (24.9 versus 23.1 mV).
Moreover, the IRCX-containing peptides dissociated readily upon
less than 30 ms of irradiation, whereas the DSS-cross-linked
peptides did not undergo efficient IRMPD upon irradiation for
250 ms, indicating that the presence of the phosphate chro-
mophore results in a much greater IR absorption at 10.6 µm (data
not shown). These collective results confirm that the successful
and selective IRMPD of the IRCX-containing peptides is due to
their high IR absorption efficiencies, not due to lower dissociation
energies.
Screening of IRCX-Cross-Linked Peptides in a Mock
Mixture by IRMPD. IRMPD analysis was performed on a mock
mixture of peptides to demonstrate selective dissociation of IRCX-
cross-linked peptides. The peptide mixture contained an equimolar
amount (10 µM) of Pyr-LYENK (1), YGGFM (2), bradykinin (3),
angiotensin II (4), R-MSH (5), Ac-RFMWMK-NH2 (6), substance
P (7), and melittin (13). Also present were equimolar amounts
(10 µM) of the IRCX intermolecular cross-linked product mixture
of R-MSH with Ac-RFMWMK-NH2 and the intramolecular cross-
linked product mixture of substance P. Products observed in the
ESI mass spectrum included R-MSH + IRCX-OH (8), R-MSH X
IRCX X R-MSH (9), substance P X IRCX (10), R-MSH X IRCX
X Ac-RFMWMK-NH2 (11), and Ac-RFMWMK-NH2 X IRCX X Ac-
RFMWMK-NH2 (12). Using direct infusion ESI-MS, the mixture
was rapidly screened by comparing the abundances of ions of m/z
450-2000 in the isolation spectrum (Supporting Information,
Figure 1A) to the IRMPD spectrum obtained using 25 ms of
irradiation at 50 W (Supporting Information, Figure 1B). The
abundances of the unmodified peptide ions are unaffected by IR
irradiation, whereas the IRCX-cross-linked peptide ions readily
photodissociate with reductions in ion abundance of ∼90% with
the exception of the singly protonated intramolecularly cross-
linked substance P ion (Supporting Information, Table 1). While
the doubly charged analogue undergoes efficient IRMPD, the
singly protonated species, which does not have a mobile proton,
4812 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

Figure 3. Product ion spectra of [R-MSH X IRCX X Ac-RFMWMK-NH2 + 3H]3+ (A) IRMPD of m/z 978.5 (q ) 0.1, 12 ms, 50 W) and (B) CID
of m/z 978.5 (q )0.25, 30 ms, 34.1 mV). R-MSH is referred to as the R-peptide and Ac-RFMWMK-NH2 as the ꢀ-peptide. The precursor ion is
represented by an asterisk (*).
doubly charged ions, leading to efficient IRMPD. No discernible
decrease in the ion abundances of the unmodified peptides was
observed using irradiation times of greater than 250 ms; this
indicates that the laser irradiation time and power has an ample
range of usability to differentiate between IRCX-cross-linked
peptides and unmodified ones.
For more complex mixtures such as those expected for
peptides with isobaric m/z values, an HPLC separation is required
prior to IRMPD screening, as demonstrated for the same mock
mixture. As detailed in the Experimental Section, the scan
sequence to accomplish this task includes acquisition of a full mass
spectrum, then an isolation mass spectrum from m/z 450 to 2000,
and then an IRMPD mass spectrum of all isolated ions. Selected
Figure 4. Time-resolved IRMPD (50 W) of [R-MSH X IRCX X Ac-
ion chromatograms (SICs) for each of the species in the mixture
were extracted and then plotted together to form a reconstructed
ion chromatogram (RIC). The reconstructed ion chromatograms
for the isolation and IRMPD spectra are shown in Figure 6, and
the changes in ion abundances are summarized in Table 1. The
difference between the RICs for the isolation and IRMPD mass
spectra is striking. Upon IR irradiation, the unmodified peptide
ions did not exhibit any decrease in ion abundance with the
exception of [melittin + 5H]5+, which decreased in abundance
by ∼20% (based on peak areas in the SICs). In contrast, the IRCX-
cross-linked and modified peptide ions are reduced on the order
of 60-100% in abundance after irradiation. All of the species that
eluted between 37 and 44 min contained either an IRCX cross-
link or an IRCX-OH modification, and upon 25 ms of IR irradiation,
RFMWMK-NH2 + 3H]3+ displaying the relative abundances of the
precursor ion (b), non-IRCX-containing product ions (9), and IRCX
containing product ions (2). Ion abundances are all relative to the
total ion abundance of the spectrum acquired with 0 ms of irradiation.
actually increased in abundance upon IR irradiation in this
experiment (relative abundance increased from 0.39 to 0.51%). This
charge-state dependence on IRMPD efficiencies provides a means
to distinguish between intra- and intermolecularly IRCX cross-
linked peptides, in which the intermolecularly cross-linked pep-
tides of all charge states exhibit high photodissociation efficien-
cies. Most intramolecularly cross-linked tryptic peptides will
possess a free N-terminus and either an unmodified Lys or Arg
at the C-terminus and thus will predominantly be observed as
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 4813

Figure 5. Product ion spectra of [substance P X IRCX + 2H]2+ (A) IRMPD of m/z 839.3 (q ) 0.15, 50 ms, 35 W) and (B) CID of m/z 839.3
(q )0.25, 30 ms, 26.7 mV). The precursor ion is represented by an asterisk (*).
Figure 6. Reconstructed ion chromatograms of (A) isolation (25 ms) and (B) IRMPD (25 ms, 50 W) spectra from LC-IRMPD-MS of a mock
mixture of peptides. The identities of the eluting species are listed in Table 2. Ion chromatogram intensities of [R-MSH + IRCX-OH + 2H]2+ (8)
and [R-MSH X IRCX X R-MSH + 4H]4+ (9) were zoomed in 10× in both (A) and (B).
the intact peptide ions containing the IRCX chromophore were
no longer detected.
dead-end modification as well as singly cross-linked ubiquitin. At
a 25:1 molar ratio, upward of two modifications were observed
but the singly cross-linked species was the most abundant.
Unmodified ubiquitin was still detected as the least abundant
species. The most abundant species observed in the full mass
spectrum of 50:1 IRCX-cross-linked ubiquitin corresponded to the
introduction of a single cross-link and a single dead-end modifica-
tion (Supporting Information, Figure 2). Also observed, but to
lesser degrees, was ubiquitin with two IRCX cross-links, as well
as ubiquitin possessing three total modifications (combination of
two cross-links and one dead-end modification or one cross-link
LC-IRMPD-MS of IRCX Cross-Linked Ubiquitin. To test
the utility of LC-IRMPD-MS for screening and identifying IRCX-
cross-linked peptides, a trypsin digest of IRCX-cross-linked ubiq-
uitin was analyzed as a model cross-linked protein. Upon incuba-
tion with IRCX, ESI-MS analysis indicated that ubiquitin was
modified to different degrees. For the lowest molar ratio of cross-
linker to protein (5:1), the most abundant species present was
unmodified ubiquitin; also observed at approximately the same
abundance in the ESI mass spectrum was ubiquitin with a single
4814 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

Table 1. Decreases in Ion Abundance upon IR Irradiation from LC-IRMPD-MS Reconstructed Ion Chromatograms of
a Mock Mixture of Peptidesa
peak
area
isolation
peak
area
IRMPD
percent
abundance
decrease
elution
(min)
label
species
m/z
1
2
3
11.8
[Pyr-LYENK + H]+
[YGGFM + H]+
777.4
574.2
2072.76
5402.80
2942.05
49.78
1853.80
6251.49
3595.30
53.84
3087.37
122.36
6994.73
1717.75
7.12
10.6
-15.7
-22.2
-8.2
20.7
20.9
[bradykinin + 2H]2+
[bradykinin + H]+
530.7
20.9
1060.5
523.8
4
5
23.5
[angiotensin II + 2H]2+
2806.80
131.89
-10.0
7.2
23.5
[angiotensin II + H]+
1046.5
555.6
26.8
[R-MSH + 3H]3+
5863.15
1533.24
5.28
7154.23
181.80
7937.77
179.17
190.39
53.83
7.89
2459.99
77.85
894.07
69.18
218.90
1165.07
19847.59
18792.54
4885.16
-19.3
-12.0
-35.0
-13.8
7.2
-19.6
-5.9
26.8
[R-MSH + 2H]2+
832.9
26.8
[R-MSH + H]+
1664.8
470.2
6
7
28.0
[Ac-RFMWMK-NH2 + 2H]2+
[Ac-RFMWMK-NH2 + H]+
[substance P + 2H]2+
8144.27
168.69
9496.41
189.83
0.00
28.0
939.5
674.4
28.9
28.9
[substance P + H]+
1347.7
1006.9
915.4
1220.2
839.4
1677.7
978.4
1467.1
736.6
1104.5
570.1
8
9
37.9
38.3
28.3
40.6, 41.4
40.6, 41.4
40.9
40.9
44.4
44.4
44.7
[r-MSH + IRCX-OH + 2H]2+
[r-MSH X IRCX X r-MSH + 4H]4+
[r-MSH X IRCX X r-MSH + 3H]3+
[substance P X IRCX + 2H]2+
[substance P X IRCX + H]+
[r-MSH X IRCX X Ac-RFMWMK-NH2 + 3H]3+
[r-MSH X IRCX X Ac-RFMWMK-NH2 + 2H]2+
[2(Ac-RFMWMK-NH2) X IRCX + 3H]3+
[2(Ac-RFMWMK-NH2) X IRCX + 2H]2+
[melittin + 5H]5+
100.0
100.0
99.2
100.0
59.8
100.0
81.4
94.7
98.9
19.2
0.00
0.06
10
11
0.06
31.29
0.00
12.89
11.68
12.79
16037.27
19778.43
5050.55
12
13
44.7
44.7
[melittin + 4H]4+
712.4
949.6
-5.2
-3.4
[melittin + 3H]3+
a Percent abundance decreases calculated from the decrease of the peak areas of the SICs from the isolation and IRMPD spectra.
Table 2. Decreases in Ion Abundance upon IR Irradiation from LC-IRMPD-MS Reconstructed Ion Chromatograms of
a Tryptic Digest of Ubiquitin Cross-Linked by IRCX
average %
elution
(min)
primary
abundance std
label
species
m/z
sequenceb
decreasec devc
1
0.6
[30-42]3+
[55-63]2+
[43-48]+
[64-72]2+
[12-27]2+
[55-72]3+
509.0 IQDKEGIPPDQQR
541.3 TLSDYNIQK
31
14
4
2
15.5
-28
3
16.6
22.0
26.0
27.0
648.3 LIFAGK
-4
5
4
534.6 ESTLHLVLR
894.6 TITLEVEPSDTIENVK
710.8 TLSDYNIQKESTLHLVLR
1036.1 IQDK*EGIPPDQQR X IRCX X TLTGK*TITLEVEPSDTIENVK
848.0 LIFAGK*QLEDGR + IRCX-OH
797.8 TLSDYNIQK*ESTLHLVLR + IRCX + IQDK*EGIPPDQQR
996.6 TLSDYNIQK*ESTLHLVLR X IRCX X IQDK*EGIPPDQQR
807.0 MQIFVK*TLTGK + IRCX-OH
826.8 TLSDYNIQK*ESTLHLVLR + IRCX-OH
762.9 TLSDYNIQK*ESTLHLVLR X IRCX X LIFAGK*QLEDGR
952.8 TLSDYNIQK*ESTLHLVLR X IRCX X LIFAGK*QLEDGR
-28
17
15
24
5
-37
6
-66
7
8
9
31.6, 33.2 [30-42 X IRCX X 7-27]4+
100.0
100.0
100.0
100.0
99
100.0
100.0
92
0.0
32.8, 35.4 [43-54 + IRCX-OH]2+
0.0
0.0
0.0
1
33.1
33.1
[55-72 X IRCX + 30-42]5+
[55-72 X IRCX X 30-42]4+
10 35.3, 37.6 [1-11 + IRCX-OH]2+
11 36.0, 37.9 [55-72 + IRCX-OH]3+
0.0
0.0
5
12 37.0
37.0
[55-72 X IRCX X 43-54]5+
[55-72 X IRCX X 43-54]4+
37.0
[55-72 X IRCX X 43-54]3+ 1269.6 TLSDYNIQK*ESTLHLVLR X IRCX X LIFAGK*QLEDGR
99.4
100.0
99.9
0.8
0.0
0.1
13 37.8
37.8
[55-72 X IRCX X 1-11]5+
[55-72 X IRCX X 1-11]4+
746.9 TLSDYNIQK*ESTLHLVLR X IRCX X MQIFVK*TLTGK
932.7 TLSDYNIQK*ESTLHLVLR X IRCX X MQIFVK*TLTGK
a Percent abundance decreases calculated from the decrease of the peak areas of the SICs from the isolation and IRMPD spectra. b Site of
modification or cross-link represented by *. c Standard deviation of the percent abundance decreases obtained in triplicate.
and two dead-end modifications). All LC-IRMPD-MS analyses were
performed on the product mixture of the 50:1 molar ratio of IRCX
to ubiquitin to eliminate any unmodified ubiquitin from the sample.
No separation of the different IRCX cross-linked and modified
ubiquitin products was performed, and all products were subjected
simultaneously to trypsin digestion. After digestion, the tryptic
peptides were separated and analyzed by LC-IRMPD-MS in a
manner similar to the mock mixture described above, acquiring
isolation and IRMPD spectra of ions of m/z 450-2000. The RICs
for the isolation spectra and IRMPD spectra are shown in Figure
7, and the eluting species are summarized in Table 2. The
differences between the two chromatograms are dramaticsonly
the IRCX-cross-linked or modified peptides (species labeled 7-13)
exhibit IRMPD, and upon 50 ms of irradiation decrease in ion
abundance to below the detection limit. On average, the unmodi-
fied peptide ions exhibited an increase in ion abundance of ∼20%,
which we attribute to fluctuations in ion trapping during the
isolation and IRMPD steps, as well as to differences in the number
of ions being injected into the trap between the two scans, despite
using the automatic gain control feature of the ion trap. The IRCX-
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 4815

Figure 7. Reconstructed ion chromatograms of (A) isolation (50 ms) and (B) IRMPD (50 ms, 50 W) spectra from LC-IRMPD-MS of a tryptic
digest of ubiquitin cross-linked by IRCX. See Table 3 for identities of eluting species. Ion chromatogram intensities of [43-48]+ (3), [55-72]3+
(6), [30-42 X IRCX X 7-27]4+ (7), [55-72 X IRCX X 30-42]5+ (9), [1-11 + IRCX-OH]2+ (10), [55-72 + IRCX-OH]3+ (11), and [55-72 X
IRCX X 1-11]5+ (13) were zoomed in by 10× in both (A) and (B).
Table 3. Summary of Cross-Links and Modifications of Ubiquitin Identified Using IRCX and LC-IRMPD-MS
complex
mass
complex
mass
charge
states
cross-linked/
modified
site(s)
tryptic
distance
(Å)c
fragment
peptide sequence(s)a
measuredb
calculatedb
observed
intensityd
0.5
30-42
7-27
IQDK*EGIPPDQQR
4140.4
3982.8
3805.7
3724.9
4140.0
3982.0
3804.9
3723.9
4+
Lys-33
Lys-11
Lys-63
Lys-33
Lys-63
Lys-48
Lys-63
Lys-6
13.3
19.6
18.4
15.1
TLTGK*TITLEVEPSDTIENVK
TLSDYNIQK*ESTLHLVLR
IQDK*EGIPPDQQR
55-72
30-42
55-72
43-54
55-72
1-11
5+, 4+
151.3
1421.0
177.3
TLSDYNIQK*ESTLHLVLR
LIFAGIK*QLEDGR
5+, 4+, 3+
5+, 4+
TLSDYNIQK*ESTLHLVLR
MQIFVK*TLTGK
43-54
1-11
LIFAGIK*QLEDGR
MQIFVK*TLTGK
1693.8
1613.3
1694.8
1613.8
2+
2+
Lys-48
Lys-6
n/a
n/a
3067.1
226.8
a Site of modification or cross-link represented by *. b Neutral monoisotopic mass in Da. c Distance between R-carbons of lysine residues calculated
from X-ray crystal structure of ubiquitin (PDB: 1AAR). d Total ion current in counts from full MS scans in isolation/IRMPD LC-IRMPD-MS experiment.
cross-linked peptides decreased by ∼100%, with the exception of
None of the unmodified tryptic peptides of ubiquitin underwent
IRMPD, again allowing for them to be distinguished from the
IRCX-cross-linked peptides. An example of an IRMPD mass
spectrum of an unmodified peptide is shown in Supporting
Information, Figure 3A, in which doubly protonated T12ITL-
EVEPSDTIENVK27 does not photodissociate upon 30 ms of IR
irradiation; the precursor ion remains the only ion observed in
the spectrum. In contrast, IRMPD spectra acquired during the
data-dependent LC-IRMPD-MS method for three IRCX-cross-
linked peptides are shown in Figure 8, and in all cases, the
precursor ions readily photodissociate. For example, the second
[T55LSDYNIQK63ESTLHLVLR72
X
IRCX
X
L43IFAGIK48
-
QLEDGR54 + 4H]4+, which was reduced in abundance by 92%.
The abundance of this quadruply charged cross-linked peptide
likely did not decrease by 100% due to an isobar with the y10ꢀ
-
173+ product ion, corresponding to cleavage C-terminal to the
cross-linked Lys-48 of L43IFAGK48QLEDGR54 and incomplete
secondary dissociation of this fragment ion. Even with this isobaric
overlap, there is a consistent difference in IRMPD efficiencies that
allows the IRCX-cross-linked peptides to be readily distinguished.
While comparing isolation and IRMPD spectra to determine
which peptide ions undergo photodissociation upon IR irradiation
allows rapid pinpointing of the IRCX-cross-linked peptides, one
cannot directly use these IRMPD spectra to locate the site of cross-
linking because all ions of m/z 450-2000 are irradiated simulta-
neously in the screening step, creating complicated, overlapping
dissociation patterns of all IRCX-cross-linked peptides. In order
to obtain product ion information of individual cross-linked
peptides, data-dependent MS/MS was utilized to isolate and
subsequently perform IRMPD on the most abundant ions.
cross-linked peptide to elute contains T55LSDYNIQK63
-
ESTLHLVLR72 (R-peptide) linked to I30QDK33EGIPPDQQR42 (ꢀ-
peptide) through Lys-63 and Lys-33. IRMPD of this cross-linked
peptide ion provided 10 unique sequence ions (Figure 8A). No
product ions containing the IRCX cross-link were observed, most
likely a result of secondary dissociation of these fragment ions.
Almost a full series of yR ions without the IRCX cross-link (e.g.,
yR ions C-terminal of the cross-linked Lys-63) were detected,
providing a sequence tag for identifying one of the two constituent
4816 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

Figure 8. IRMPD spectra (30 ms, 50 W, q )0.1) of tryptic peptides of ubiquitin cross-linked by IRCX acquired during data-dependent LC-
IRMPD-MS experiment: (A) [T55LSDYNIQK63ESTLHLVLR72
X IRCX X +
I30QDK33EGIPPDQQR42 5H]5+ of m/z 797.8, (B)
[T55LSDYNIQK63ESTLHLVLR72 X IRCX X M1QIFVK6TLTGK11 + 5H]5+ of m/z 746.1, and (C) [T55LSDYNIQK63ESTLHLVLR72 X IRCX X
L43IFAGIK48QLEDGR54 + 5H]5+ of m/z 762.4. The first peptide listed is the R-peptide; the second peptide is referred to as the ꢀ-peptide.
Lysine residues in boldface font indicate the site of the cross-link. Precursor ions, if observed, are labeled with an asterisk (*).
peptides. The highly abundant y6ꢀ ions, result of cleavage N-
terminal to Pro-37, provide information necessary to determine
that the ꢀ-peptide contains P37PDQQR42. However, the corre-
sponding b7ꢀ product ion, which contains the chromophore and
the R-peptide, can undergo secondary dissociation to yield
additional diagnostic fragment ions. Along with the identity of the
R-peptide and the intact mass of the cross-linked peptide, one can
deduce the identity of the second peptide and finally the location
of the cross-link, between Lys-63 and Lys-33. In contrast, CID of
the 5+ charge state of this cross-linked peptide yielded only seven
4+
identifiable product ions (y6ꢀ2+, y6ꢀ+, y8R2+, y4ꢀ+, y8ꢀ+, y16R
,
b7ꢀ3+), three of which correspond to cleavage N-terminal to Pro-
37 (Supporting Information, Figure 3B). No sequential series of
product ions was observed using CID, and most of the ions
observed could not be identified as typical a-, b-, or y-type ions
(including double-cleavage products). The IRCX-cross-linked pep-
tide containing T55LSDYNIQK63ESTLHLVLR72 (R-peptide) and
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 4817

M1QIFVK6TLTGK11 (ꢀ-peptide) readily photodissociated upon IR
irradiation, yielding only fragment ions without the IRCX chro-
mophore (Figure 8B). Thirteen of the 14 amide bonds C-terminal
to the cross-linked lysines were cleaved by IRMPD, providing a
sequence tag for both constituent peptides. CID of this cross-
linked peptide yielded a variety of product ions including both b-
and y-type fragment ions with and without the cross-link (Sup-
porting Information, Figure 3C). In addition several double-
marized in Table 3. IRCX has a spacer arm length of ∼16 Å, and
given the flexibility of lysine side chains, distances of ∼28 Å
between R-carbons of lysines could be spanned. For all four of
the observed cross-links, the distance between the R-carbons was
less than 20 Å based on the X-ray crystal structure (PDB: 1AAR).
The two phenyl groups of IRCX create a more rigid cross-linker
that may not be able to easily span short distances, as evident by
the fact that no cross-links spanning a distance of less than 13 Å
were observed. The rigidity of IRCX would not promote the
formation of short cross-links as the one observed between Lys-6
and Lys-11 (∆x ) 5.8 Å) in a previous study using DSS.54 Three
of the four cross-links identified in the present study involved Lys-
63, which is located on the surface of ubiquitin. Upon creation of
an amide bond between IRCX and Lys-63, the other NHS-ester of
IRCX could react with other surface-accessible lysines within 20
Å, including Lys-48, Lys-33, and Lys-6, yielding a mixture of cross-
linked products, as observed. While other lysines are potential
sites of cross-linking, Lys-63 is likely more reactive resulting in
limited structural information. Recently, a partial acetylation
technique has been used to enhance the diversity of cross-linking
reactions by blocking the more reactive lysine residues to provide
more comprehensive distance constraints.55 Using such an ap-
proach may yield more informative cross-links between other
lysine pairs. The two dead-end modifications were located at Lys-
48 and Lys-6, two other surface-accessible primary amines that
were also observed to be cross-linked.
L
cleavage product ions were observed including a series of y16R ynꢀ
ions (i.e., y16R fragment linked to a y-ion of the ꢀ-peptide), which
complicate interpretation of the CID spectrum. IRMPD alone
provides far more diagnostic product ions allowing for the cross-
linked peptide to be identified. For the cross-linked peptide ion
[T55LSDYNIQK63ESTLHLVLR72
X
IRCX
X
L43IFAGK48
-
QLEDGR54 + 5H]5+, all of y-ions C-terminal to the two cross-
linked lysine residues were detected using IRMPD (Figure 8C).
Using these y-ions, one could easily sequence the C-terminal ends
of the two constituent peptides via a de novo approach. In addition,
three bnꢀ product ions were detected providing confirmation of
the identity of the ꢀ-peptide, L43IFAGIK48QLEDGR54. Two dead-
end modifications, at Lys-48 and Lys-6, could also be identified.
The IRMPD mass spectrum for the species containing
L43IFAGIK48QLEDGR54 modified by IRCX-OH at Lys-48 is shown
in Supporting Information, Figure 3D. Cleavage of all backbone
amide bonds occurred, yielding a series of b-ions N-terminal to
the modified lysine and a series of y-ions C-terminal to the
modified lysine, all without the IRCX chromophore.
A majority of the product ions observed upon IRMPD were
y-ions without the IRCX cross-link, as opposed to b-ions. Earlier
work in our laboratory suggested that there is a preference for
cleavage C-terminal to the cross-linked lysine for intermolecularly
cross-linked peptides, possibly due to charge localization on the
C-terminal arginine residues.32 Dissociation at this amide bond
of the R-peptide would result in a yR-ion with a protonated
C-terminal arginine and a bR-ion retaining the IRCX cross-link and
the ꢀ-peptide. This bR-ion would likely undergo secondary and
higher order dissociation since it contains the phosphate chro-
mophore, possibly producing an un-cross-linked yꢀ-ion. Any
secondary product ion still retaining the chromophore may
continue to absorb photons and dissociate, resulting in small
fragments that are not observed (e.g., neutral fragments, m/z
below the low-mass cutoff, etc.). However, if a charge is retained
on the N-terminus, un-cross-linked b-ions would also be detected.
In all of the IRMPD mass spectra of the IRCX-cross-linked and
IRCX-OH modified peptides, a product ion of m/z 121 was
detected. This fragment ion, observed only after significant
secondary dissociation, corresponds in mass to a double-cleavage
productscleavage of the cross-link amide bond and between the
P-O bond nearest the cleaved amide bond. The formation of this
diagnostic product ion, which is not isobaric with any immonium
ions of the amino acids, provides another means of distinguishing
IRCX-cross-linked peptides from unmodified ones. Using tradi-
tional CID, one would not observe this reporter ion as it has an
m/z value below the low-mass cutoff. Moreover, as a higher-order
product ion, it is only observed after significant secondary
dissociation afforded by IRMPD, not CID.
CONCLUSIONS
By incorporating a phosphate chromophore into our chemical
cross-linker IRCX, we have shown that both intra- and intermo-
lecularly cross-linked peptides can be rapidly distinguished from
unmodified peptides using IRMPD-MS. Using 50 ms of irradiation,
only the IRCX cross-linked or dead-end modified peptides under-
went efficient photodissociation, thus allowing the identification
of these key species by simply comparing ion abundances before
and after exposure to IR irradiation. This technique has been
successfully applied to both direct-infusion ESI-IRMPD-MS and
online LC-IRMPD-MS using data-dependent MS/MS experiments
for a mock mixture of peptides and a tryptic digest of IRCX-cross-
linked ubiquitin. For the cross-linked ubiquitin sample, a total of
four cross-links and two dead-end modifications were observed,
and the locations of the cross-links were unambiguously identified.
IRMPD of the intermolecularly cross-linked peptides yielded a
series of y-ions C-terminal to the cross-linked lysines without the
IRCX cross-link due to secondary dissociation of fragment ions
containing the chromophore. These product ions could then be
used to sequence each of the two constituent peptides in a de
novo manner, potentially providing a ready means to identify not
only the cross-linked peptides but also the proteins in an
intracellular chemical cross-linking experiment.
ACKNOWLEDGMENT
Funding from the Welch Foundation (F1155) and the National
Science Foundation (CHE-0718320) is gratefully acknowledged.
(54) Kruppa, G. H.; Schoeniger, J.; Young, M. M. Rapid Commun. Mass Spectrom.
2003, 17, 155–162
(55) Guo, X.; Bandyopadhyay, P.; Schilling, B.; Young, M. M.; Fujii, N.; Aynechi,
T.; Guy, R. K.; Kuntz, I. D.; Gibson, B. W. Anal. Chem. 2008, 80, 951–960
.
In total, four cross-links and two dead-end modifications were
identified using the IRCX and LC-IRMPD-MS strategy, as sum-
.
4818 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

ThermoFisher is also gratefully acknowledged for their assistance
in the modification of the instrument control files.
1-11]5+, and IRMPD of [43-54 + IRCX-OH]2+; Table 1, relative
ion abundance decreases upon IR irradiation for the mock mixture
of peptides. This material is available free of charge via the Internet
at http://pubs.acs.org.
SUPPORTING INFORMATION AVAILABLE
Figure 1, isolation and IRMPD spectra of a mock mixture of
peptides; Figure 2, deconvoluted and full ESI-MS of IRCX cross-
linked ubiquitin; Figure 3, product ion spectra of tryptic peptides
Received for review March 27, 2008. Accepted May 13,
2008.
of IRCX cross-linked ubiquitin including IRMPD of [12-27]2+
,
CID of [55-72 X IRCX X 30-42]5+, CID of [55-72 X IRCX X
AC800625X
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 4819