Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry

Article abstract of DOI:10.1016/j.jasms.2010.04.004

Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function in various cellular processes. Chemical cross-linking coupled with mass spectrometry has proven to be a powerful method not only for mapping protein-prote

Full text of DOI:10.1016/j.jasms.2010.04.004

Selective Enrichment and Identification of
Azide-tagged Cross-Linked Peptides Using
Chemical Ligation and Mass Spectrometry
Danielle Vellucci,a Athit Kao,b Robyn M. Kaake,b Scott D. Rychnovsky,a
and Lan Huangb
a Department of Chemistry, University of California, Irvine, California, USA
b Departments of Physiology and Biophysics and Developmental and Cell Biology, University of California,
Irvine, California, USA
Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function
in various cellular processes. Chemical cross-linking coupled with mass spectrometry has proven
to be a powerful method not only for mapping protein-protein interactions of all natures, including
weak and transient ones, but also for determining their interaction interfaces. One critical challenge
remaining in this approach is how to effectively isolate and identify cross-linked products from a
complex peptide mixture. In this work, we have developed a novel strategy using conjugation
chemistry for selective enrichment of cross-linked products. An azide-tagged cross-linker along
with two biotinylated conjugation reagents were designed and synthesized. Cross-linking of
model peptides and cytochrome c as well as enrichment of the resulting cross-linked peptides has
been assessed. Selective conjugation of azide-tagged cross-linked peptides has been demonstrated
using two strategies: copper catalyzed cycloaddition and Staudinger ligation. While both methods
are effective, Staudinger ligation is better suited for enriching the cross-linked peptides since there
are fewer issues with sample handling. LC MSn analysis coupled with database searching using the
Protein Prospector software package allowed identification of 58 cytochrome c cross-linked
peptides after enrichment and affinity purification. The new enrichment strategy developed in this
work provides useful tools for facilitating identification of cross-linked peptides in a peptide
mixture by MS, thus presenting a step forward in future studies of protein-protein interactions of
protein complexes by cross-linking and mass spectrometry. (J Am Soc Mass Spectrom 2010, 21,
1432–1445) © 2010 American Society for Mass Spectrometry
roteins form stable and/or dynamic multi-
subunit protein complexes under different phys-
iologic conditions to maintain cell viability and
spectroscopy requires large quantities of pure protein in a
specific solvent, and X-ray crystallography is often limited
by the crystallization process. The combination of chemi-
cal cross-linking, enzymatic digestion, and subsequent
mass spectrometric analysis for the elucidation of three-
dimensional protein structures offers distinct advantages
over these traditional methods due to its speed, sensitiv-
ity, and versatility [2–4]. This strategy has been success-
fully employed for unraveling protein complex topology
and protein-protein interacting interfaces [7–17, 26].
Due to the inherent complexity in cross-linking reac-
tions and the abundance of non-cross-linked peptides,
detection of cross-linked peptides in a complex mixture is
quite challenging [2, 4]. The digested mixture of cross-
linked protein complexes typically contains four types of
peptides: (1) unmodified peptides of the complex compo-
nents, (2) dead end modified peptides, (3) intramolecular
(inter-linked and intra-linked) cross-linking products be-
tween peptides originated from one protein, and (4)
intermolecular cross-linking products (inter-linked) be-
tween peptides from different proteins [3, 8]. To facilitate
the identification process, various methods using biotin-
ylated [27–31], isotope-labeled [32–34], fluorescently la-
beled [35, 36], or mass-tag labeled cross-linking reagents
P
normal cell homeostasis. A thorough understanding of
protein interactions and structures of protein complexes is
fundamental to the understanding of protein function and
regulation. Chemical cross-linking coupled with mass
spectrometry (MS) is a powerful method that can be used
to study protein-protein interactions [1–6]. The unique
capability of chemical cross-linking to stabilize protein
interactions through covalent bond formation allows not
only the structural elucidation [7–17], but also the detec-
tion of stable, weak, and/or transient protein-protein
interactions in native cells or tissues [18–25].
In addition to capturing protein interacting partners,
many studies have shown that chemical cross-linking can
yield low-resolution structural information about the con-
straints within a molecule [2–4]. Traditional methods such
as NMR analysis and X-ray crystallography can yield
detailed information on protein structure, however NMR
Address reprint requests to Dr. L. Huang, Medical Science I, D233,
Department of Physiology and Biophysics, Department of Developmental
and Cell Biology, University of California, Irvine, CA 92697-4560, USA.
E-mail: lanhuang@uci.edu
Published online April 24, 2010
© 2010 American Society for Mass Spectrometry. Published by Elsevier Inc.
1044-0305/10/$32.00
doi:10.1016/j.jasms.2010.04.004
Received February 20, 2010
Revised April 10, 2010
Accepted April 11, 2010

J Am Soc Mass Spectrom 2010, 21, 1432–1445
ENRICHMENT OF CROSS-LINKED PEPTIDES 1433
[28, 37] have been reported. Among them, biotinylated
cross-linkers are advantageous due to their specificity and
efficiency. However, these linkers have the drawback of
being bulky and therefore not very effective or ideal for in
vivo protein cross-linking [4, 38]. Recently, new strategies
have also been developed based on click chemistry with
chemical conjugation of azide and alkyne groups using
alkyne-tagged [39] or azide tagged [40, 41] cross-linkers.
Applications using click chemistry for enrichment of other
types of biomolecules have also been reported [42–49].
The marked advantage of this approach is the complete
orthogonality to functional groups in normal biomol-
ecules since azides and alkynes are not present in pep-
tides, proteins, nucleic acids, or polysaccharides, and are
very rare in all other biological materials and natural
products. With the absence of significant competing reac-
tions [43], click chemistry shows great promise for protein
cross-linking studies.
myelin peptide (Ac-ASQKRPSQRHG, 92.7% purity) was
purchased from American Peptide (Sunnyvale, CA) and
Ac-IR7 peptide (Ac-IEAEKGR, 98.1% purity) was synthe-
sized by GL Biochem (Shanghai, China).
Synthesis and Characterization of Azide-DSG,
Biotin-Alkyne, and Biotin-Phosphine
The synthesis schemes for azide-DSG (S-5) (spacer length
7.7 Å), biotin-alkyne (S-11), and biotin-phosphine (S-17)
are displayed in Figure 1. As shown, azide-DSG was
synthesized from commercially available dimethyl 1,3-
acetonedicaboxylate (S-1) in four steps. Biotin-alkyne was
synthesized from S-6 in five steps. Biotin-phosphine was
synthesized from S-12 in three steps based on Bertozzi’s
protocols [53, 63]. The details in synthesis and chemical
characterization of each product have been summarized
in Supplemental methods, which can be found in the
electronic version of this article.
Although both alkyne-tagged [39] and azide-tagged
cross-linkers [41] can be used for the same purpose,
azide-tagged reagents have some advantages that
would be preferred. Azides present a high chemical
stability under physiologic conditions and unlike alkynes,
their reactivity enables several types of mild and selective
organic transformations, including Cu(I)-catalyzed [3 ϩ 2]
cycloaddition [42, 50], strain-promoted [3 ϩ 2]-cycloaddition
[49, 51, 52], and Staudinger ligation [53–55]. The
Staudinger ligation has been widely used in biological
applications, especially for selective enrichment of chemi-
cally modified peptides [56–61], but its applicability in
the study of cross-linked peptides has not been evalu-
ated. Due to complications in sample handling using
copper-catalyzed click chemistry and challenges in syn-
thesizing strained alkynes, the alternative strategy us-
ing Staudinger ligation may be advantageous for isola-
tion of cross-linked peptides. In this work, we have
explored azide-based conjugation chemistry using
Staudinger ligation and developed a new enrichment
strategy for cross-linked peptides using a newly de-
signed azide-tagged cross-linker and biotin-phosphine.
These new reagents not only allow efficient cross-linking
of model peptides and cytochrome c and subsequent
enrichment of cross-linked peptides, but also provide
diagnostic ions in MS/MS analysis to facilitate MS iden-
tification of cross-linked peptides with high confidence. In
combination with various software tools (i.e., Batch-tag,
MS-bridge, MS-product and Search compare) in Protein
Prospector [62], LC MS/MS analyses allowed identifica-
tion of 58 enriched cytochrome c cross-linked peptides.
Cross-Linking of Synthetic Peptides with
Azide-DSG
A solution of tryptic-like synthetic peptides Ac-myelin
or Ac-IR7 (0.31 ␮mol) in DMF (400 ␮L) was mixed with
20 ␮l 8 mmol/L azide-DSG cross-linker in DMF, and
the reaction mixture was treated with 20 ␮L 8 mmol/L
diisopropylethylamine in DMF. The reaction mixture
was stirred for 16 h at ambient temperature. The solu-
tion was then dried by SpeedVac and resuspended in
PBS buffer or 20% ACN.
Cross-Linking of Cytochrome c with Azide-DSG
Two hundred ␮M bovine cytochrome c in 1X PBS (pH
7.5) was reacted with azide-DSG in DMSO at 20:1
(linker:protein) molar ratio and incubated at room tem-
perature for 1 h with agitation. After quenching with
glycine, it was then digested with 1% trypsin (wt/wt)
overnight at 37 °C before enrichment.
Enrichment of Azide-Tagged Cross-Linked Peptides
Using Copper Catalyzed Click Chemistry
A solution of azide-DSG cross-linked Ac-myelin pep-
tide (20 ␮L, 2.6 ϫ 10Ϫ8 mol) in PBS buffer was treated
with 20 ␮L of a 13 mM solution of biotin-alkyne in DMSO.
Subsequently, 40 ␮L of 6.5 mM CuSO4·5H2O, 4 ␮L of 193
mM sodium ascorbate, and 8 ␮L of 6.3 mM TBTA solution
were added [64]. The reaction mixture was gently stirred
for 18 h. The solution was then desalted by a C18 ZipTip
(Varian, Palo Alto, CA, USA), dried by SpeedVac, and
resuspended in 1.2 mL of 4% ACN/0.1% formic acid.
Following desalting, 5 ␮L of the mixture was incubated
with 400 ␮L of Dynabeads M-280 streptavidin (Invitrogen,
Carlsbad, CA, USA). After 30 min incubation, the beads
were washed with PBS buffer and 20% ACN. Peptides
were eluted from the beads with 90% TFA. The TFA
Materials and Methods
Materials and Reagents
All general chemicals for buffers were purchased from
Fisher Scientific, now owned by ThermoFisher Scientific,
Waltham, MA, USA or VWR International, West Chester,
PA, USA. Bovine heart cytochrome c (98% purity) was
purchased from Sigma-Aldrich, St. Louis, MI, USA. Ac-

1434 VELLUCCI ET AL.
J Am Soc Mass Spectrom 2010, 21, 1432–1445
Figure 1. Synthetic schemes of (a) azide-DSG (S-5); (b) biotin-alkyne (S-11); (c) biotin-phosphine (S-17).
solution was dried by SpeedVac and resuspended in 4%
ACN/0.1% formic acid for analysis.
cross-linked Ac-myelin peptide (7.73 ϫ 10Ϫ9 mol) was
treated with 7 ␮L of a 25 mM stock solution of biotin-
phosphine (S-17 in Figure 1) in DMSO. The reaction
mixture was heated to 60 °C. After 16 h, 1.5 ␮L of the
reaction mixture was incubated with 200 ␮L of Dyna-
beads M-280 streptavidin (Invitrogen). After 30 min, the
beads were washed with 200 ␮L of 20% ACN, 200 ␮L
of PBS buffer, and 200 ␮L of 25% isopropanol. The
Enrichment of Azide-Tagged Cross-Linked Peptides
Using Staudinger Ligation
Staudinger ligation was performed with a similar pro-
cedure as described [53]. Briefly, 10 ␮L of azide-DSG

J Am Soc Mass Spectrom 2010, 21, 1432–1445
ENRICHMENT OF CROSS-LINKED PEPTIDES 1435
peptides were then eluted with 85% TFA. The TFA
solution was dried by SpeedVac and the sample was
resuspended in 4% ACN/0.1% formic acid for LC MS
analysis.
version of glutamine to pyroglutamic acid. For all of the
ions being sequenced in MS/MS, their parent monoiso-
topic masses and charges were extracted from Search
Compare and submitted to MS-bridge program to iden-
tify putative cross-linked peptides by mass fitting
against theoretical masses of cross-linked cytochrome c
peptides with the given cross-linker. The parent mass
error for MS-bridge search was set as Ϯ20 ppm and
only one bridge was allowed in the cross-linked pep-
tides. All of the three types of cross-linked peptides [8],
i.e., inter-linked (type 2), intra-linked (type 1), and
dead-end modified (type 0) peptides, can be computed
and matched in MS-bridge. MS-tag was used to identify
the additional peptide sequence in the inter-linked
peptides, and MS-product was applied to validate the
identified sequences. Due to difficulties in the analysis
of the complex fragmentation of cross-linked peptides
LC MSn Analysis
LC MSn analysis of cross-linked peptides was carried
out using an LTQ-Orbitrap XL MS (Thermo Scientific,
San Jose, CA, USA) coupled with an Eksigent NanoLC
system (Eksigent, Dublin, CA, USA). The LC analysis
was performed using a capillary column (100 ␮m i.d.
ϫ150 mm long) packed with C18 resins (GL Sciences,
Torrance, CA, USA) and the peptides were eluted using
a linear gradient of 2%–40% B in 35 min; (Solvent A:
100% H2O/0.1% formic acid; Solvent B: 100% ACN/
0.1% formic acid). For LC MS/MS analysis, a cycle of
one full FT MS scan mass spectrum (350–1800 m/z,
resolution of 60,000 at m/z 400) was followed by 10
data-dependent MS/MS acquired in the linear ion trap
with normalized collision energy (setting of 35%). Tar-
get ions selected for MS/MS were dynamically ex-
cluded for 30 s. For LC MS3 analysis, one full FT MS
scan was followed by two cycles of one MS/MS scan
and three MS3 scans acquired in the linear ion trap. MS3
scan was operated in data-dependent mode to allow
sequencing the top three most abundant fragment ions
observed in a MS/MS spectrum with exclusion of 447.2,
508.2, and 520.2 ions, which are diagnostic ions of
cross-linked peptides due to fragmentation in the con-
jugated cross-linker region in MS/MS.
by automated database searching, MS3
spectra interpre-
tation was performed manually.
Results and Discussion
Selective Isolation of Cross-Linked Peptides
In this work, a new cross-linking and enrichment strat-
egy was developed to enable effective isolation of
cross-linked peptides from a complex mixture. Figure 2
shows the overview of the cross-linking reaction and
enrichment protocols. A novel azide-tagged NHS ester-
based cross-linker (i.e., azide-DSG) was designed and
synthesized (S-5 in Figure 1a) such that the azide tag
could be used as a chemical handle to allow selective
isolation of azide-tagged cross-linked peptides using
azide-based conjugation chemistry including azide-
alkyne cycloaddition and Staudinger ligation chemistry
[42, 49–55]. Azide-DSG was chosen for this work due to
its desirable water solubility, spacer length, membrane
permeability, expected stability, and ease of synthesis.
To determine the chemistry best suited for enrichment
of cross-linked peptides from complex mixtures, we
have designed and synthesized two new biotinylated
conjugation reagents (S-11 and S-17 in Figure 1a and c)
for enrichment strategies based on either copper cata-
lyzed click chemistry (Figure 2c) or Staudinger ligation
(Figure 2d), and their effectiveness has been evaluated
using model peptides as described below.
Identification of Cross-Linked Peptides by Database
Searching
Monoisotopic masses of parent ions and corresponding
fragment ions, parent ion charge states and ion intensi-
ties from LC MS/MS spectra were extracted using
in-house software based on Raw_Extract script from
Xcalibur ver, 2.4 (Thermo Scientific). Database search-
ing was performed with a developmental version of
Protein Prospector (ver. 5.3.2., http://proteinprospector.
ucsf.edu) using its software suite, i.e., Batch-tag,
MS-bridge, MS-product, MS-tag and Search Compare,
similarly as described [62]. It has been shown that
Batch-tag with mass modification search can be applied
for identifying cross-linked peptides [62]. Therefore, the
MS/MS data were first searched using Batch-tag with
mass modification against bovine cytochrome c se-
quence (SwissProt accession no. P62894). Mass modifi-
cation up to 4000 Da was set on uncleaved lysine
residues and protein free N-terminus. The mass accu-
racy for parent ions and fragment ions were set as Ϯ0.25
and 0.8 Da, respectively. The other search parameters
included trypsin as the enzyme, two maximum missed
cleavages, and three maximum variable modifications.
Variable modifications chosen were protein N-terminal
acetylation, methionine oxidation, and N-terminal con-
1. Enrichment of azide-tagged cross-linked peptides using
copper catalyzed click chemistry. Click chemistry-based
strategy (Figure 2c) was first evaluated using a model
peptide Ac-myelin. Cross-linking of Ac-myelin was
carried out in organic solvents with about equal
molar amount of azide-DSG and the resulting
products were analyzed by LC MS/MS. The cross-
linking reaction appeared to be highly effective as
no unmodified peptide was detected in MS spectra
after the reaction. Only two types of cross-linking
products, i.e., inter-linked (type 2) and dead-end
(type 0) modified peptides, were detected with the

1436 VELLUCCI ET AL.
J Am Soc Mass Spectrom 2010, 21, 1432–1445
Figure 2. Schematic overview of general cross-linking and enrichment strategies. (a) Cross-linking of
a peptide using azide-DSG; (b) cartoon structures of biotin-alkyne and biotin-phosphine (see detailed
structures in Figure 1); Enrichment strategies based on (c) copper catalyzed click chemistry; (d)
Staudinger ligation.
inter-linked product as the major product under our
experimental conditions. This can be attributed to
N-acetylation of Ac-myelin, one lysine residue in
the sequence, and the limited hydrolysis of the
cross-linker. The structural illustration and MS spec-
tra of the azide-tagged cross-linked products of
Ac-myelin are displayed in Figure 3a–c. A series of
ions ([M ϩ 6H]6ϩ 467.07, [M ϩ 5H]5ϩ 560.29, [M ϩ
4H]4ϩ 700.11) were detected as the Ac-myelin ho-
modimer and a triply charged ion ([M ϩ 3H]3ϩ
508.26) was measured as the dead-end modified
Ac-myelin. The mass addition to the cross-linked
peptides was 211 Da for the cross-link (inter- or
intra-link) and 229 Da for the dead-end modifica-
tion. To isolate the cross-linked peptides, the azide-
tagged cross-linked Ac-myelin products were sub-
jected to conjugation with a biotin-alkyne (S-11)
(Figure 1b). An acid cleavable site was incorporated
into the new biotin-alkyne to allow subsequent
elution after streptavidin-biotin binding during af-
finity purification of enriched cross-linked peptides.
Upon cycloaddition, the conjugated products con-
taining a triazole was formed for both inter-linked
and dead-end modified Ac-myelin peptides and
measured by MS analysis as displayed in Figure
3d–f. The azide-alkyne cycloaddition reaction was
very efficient since no detectable non-conjugated
cross-linked peptides were detected. After conjuga-
tion, the cycloaddition products were isolated by
binding to magnetic streptavidin beads followed by
TFA cleavage (Figure 2c). The structural illustration
and MS spectra of final Ac-myelin cross-linked
products are shown in Figure 3g–i. A series of ions
([M ϩ 6H]6ϩ 490.28, [M ϩ 5H]5ϩ 588.13, [M ϩ 4H]4ϩ
734.92) were observed for inter-linked Ac-myelin,
3ϩ
and a triply charged ion (MH3 554.65) was de-
tected for the dead-end product. Although isolation
of azide-tagged Ac-myelin cross-linked products
using copper catalyzed click chemistry was success-
ful, we found that addition of CuSO4 and sodium
ascorbate to the reaction mixture often caused pep-
tide precipitation, leading to problems in sample
handling and sample losses. In addition, it has been
noted that copper (I) may induce peptide oxidation
and thus hamper identification of the cross-linked
peptides by MS [41].
2. Enrichment of azide tagged cross-linked peptides using
Staudinger ligation. To avoid the problems associated
with copper catalyzed click reaction, we have inves-
tigated a strategy based on Staudinger ligation us-
ing a biotin-phosphine with an acid cleavage site
(S-17) (Figure 1c). The azide-DSG cross-linked Ac-
myelin products were then subjected to Staudinger
ligation and the resulting conjugates were affinity
purified by binding to magnetic streptavidin beads
followed by TFA cleavage. The products at each
step were monitored by LC MS/MS. Optimization
of reaction temperature was carried out by compar-
ing LC MS signal intensity of the cross-linked and
non-cross-linked Ac-myelin before and after the

J Am Soc Mass Spectrom 2010, 21, 1432–1445
ENRICHMENT OF CROSS-LINKED PEPTIDES 1437
Figure 3. Enrichment of azide-DSG cross-linked Ac-myelin peptides by chemical conjugation. (a)–(c)
Cross-linked products of Ac-myelin before enrichment: (a) structural illustration of cross-linked
peptides, R represents either an additional peptide for inter-linked or a hydroxyl group for a dead-end
modified Ac-myelin peptides; MS spectra of (b) inter-link (observed: [M ϩ 6H]6ϩ 467.07, [M ϩ 5H]5ϩ
560.29, [M ϩ 4H]4ϩ 700.11; theoretical: [M ϩ 6H]6ϩ 467.07, [M ϩ 5H]5ϩ 560.29, [M ϩ 4H]4ϩ 700.11);
(c) dead-end (observed: [M ϩ 3H]3ϩ 508.26; theoretical: [M ϩ 3H]3ϩ 508.25) modified Ac-myelin.
(d)-(i) Cross-linked products of Ac-myelin after copper catalyzed click chemistry (d)-(f), and affinity
purification/TFA cleavage (h)-(i): (d) structural illustration of conjugated products after click
chemistry; MS spectra of (e) inter-linked (observed: [M ϩ 6H]6ϩ 594.48, [M ϩ 5H]5ϩ 713.18, [M ϩ
4H]4ϩ 891.21; theoretical: [M ϩ 6H]6ϩ 594.47, [M ϩ 5H]5ϩ 713.16, [M ϩ 4H]4ϩ 891.20;) and (f) dead-end
(observed: [M ϩ 3H]3ϩ 763.06; theoretical: [M ϩ 3H]3ϩ 763.05) products; (g) structural illustration of
final cross-linked products after affinity purification and TFA cleavage; MS spectra of (h) inter-linked
(observed: [M ϩ 6H]6ϩ 490.28, [M ϩ 5H]5ϩ 588.13, [M ϩ 4H]4ϩ 734.92; theoretical: [M ϩ 6H]6ϩ 490.26,
[M ϩ 5H]5ϩ 588.11, [M ϩ 4H]4ϩ 734.88;) and (i) dead-end (observed: [M ϩ 3H]3ϩ 554.65; theoretical:
[M ϩ 3H]3ϩ 554.62) modified peptides. (j)–(o) Cross-linked products of Ac-myelin after Staudinger
ligation (j)-(l) and affinity purification/TFA cleavage (m)-(o): (j) structural illustration of Staudinger
ligation conjugates; MS spectra of (e) inter-linked (observed: [M ϩ 6H]6ϩ 609.96, [M ϩ 5H]5ϩ 728.16,
[M ϩ 4H]4ϩ 909.94; theoretical: [M ϩ 6H]6ϩ 609.96, [M ϩ 5H]5ϩ 728.15, [M ϩ 4H]4ϩ 909.94) and (f)
dead-end (observed: [M ϩ 3H]3ϩ 788.03; theoretical: [M ϩ 3H]3ϩ 788.03) products; (m) structural
illustration of the final cross-linked peptides after affinity purification and TFA cleavage; MS spectra
of (n) inter-linked (observed: [M ϩ 6H]6ϩ 537.11, [M ϩ 5H]5ϩ 644.33, [M ϩ 4H]4ϩ 805.15; theoretical:
[M ϩ 6H]6ϩ 537.10, [M ϩ 5H]5ϩ 644.32, [M ϩ 4H]4ϩ 805.15) and (o) dead-end (observed: [M ϩ 4H]4ϩ
486.49; theoretical: [M ϩ 4H]4ϩ 486.49) modified peptides. Note: (e), (f), (h), and (i) were acquired by
QSTAR MS. The rest of MS spectra were acquired by LTQ-Orbitrap XL MS.
reaction. All of the cross-linked Ac-myelin can be
conjugated with biotin-phosphine even at room
temperature since no unconjugated Ac-myelin
cross-linking product was detected, similar to that
of copper catalyzed click reaction. However the
conversion rate appears to be much faster at ele-
vated temperature (ϳ60 °C), and this could be ad-
vantageous for real protein samples with limited
concentrations to obtain efficient conjugation. The
structural illustrations and MS spectra of Staudinger
products of cross-linked Ac-myelin before and after
affinity purification/TFA cleavage are displayed in
Figure 3j–o. As shown, a series of multiply charged
ions ([M ϩ 6H]6ϩ 606.96, [M ϩ 5H]5ϩ 728.16, [M ϩ
4H]4ϩ, 909.94) were detected as the conjugate of the
inter-linked Ac-myelin (Figure 3k). TFA cleavage of
the affinity purified conjugates resulted in a loss of
419.15 Da, and the final product of inter-link Ac-
myelin was measured as [M ϩ 6H]6ϩ 537.11, [M ϩ
5H]5ϩ 644.33, and [M ϩ 4H]4ϩ 805.15 (Figure 3n).
Similarly, Staudinger products of the dead-end Ac-
myelin before and after TFA cleavage were detected
as [M ϩ 3H]3ϩ 788.03 (Figure 3l) and [M ϩ 4H]4ϩ
486.49 (Figure 3o), respectively. The final mass ad-
dition on cross-linked peptides attributed to the
conjugated cross-linker after TFA cleavage are

1438 VELLUCCI ET AL.
J Am Soc Mass Spectrom 2010, 21, 1432–1445
649.26 Da (i.e., C34H40N3O8P) for dead-end modifi-
cation, and 631.24 Da (i.e., C34H38N3O7P) for both
intra-linked and inter-linked ones.
Similar experiments were carried out using another
model peptide Ac-IR7 in organic solvent to limit cross-
linker hydrolysis. Cross-linking of the Ac-IR7 peptide,
selective enrichment and release of the cross-linked
Ac-IR7 peptides were as efficient as Ac-myelin (Supple-
mental Figure 1). In comparison with the copper cata-
lyzed click chemistry, this approach has similar conver-
sion efficiency, but precipitation can be completely
avoided and background reactions were largely sup-
pressed. Therefore, the Staudinger ligation has been se-
lected as the method for enrichment of azide-containing
cross-linked peptides in the sections described below.
Capture of a Cross-Linked Model Peptide from a
Protein Digest Mixture
To test the effectiveness of the enrichment of azide-DSG
cross-linked peptides in a mixture, we mixed azide-
DSG cross-linked Ac-myelin with chicken lysozyme
tryptic digest in a 1:1 ratio. The peptide mixture was
subjected to Staudinger conjugation, then affinity puri-
fied and eluted by TFA. This process was monitored by
LC MS analysis at each step as seen in Figure 4. As
shown, the Ac-myelin cross-linked peptides (i.e., inter-
linked and dead-end) were not the most abundant
species either before (Figure 4a) or after Staudinger
ligation (Figure 4b). However, after affinity purification
and acid cleavage elution, both inter-linked and dead-
end products were successfully isolated and the inter-
linked Ac-myelin became the second most abundant
signal in the purified sample (Figure 4c). This result
demonstrates the effectiveness of the selective isolation
of azide-tagged cross-linked peptides from a peptide
mixture. Due to the presence of the acid cleavage site in
the biotin-phosphine, the excess biotin-phosphine used
in the reaction that bound to the streptavidin beads was
also present in the final elution, thus leading to unde-
sirable signals, including the most abundant peak (la-
beled with an asterisk “*”) in the sample (Figure 4c).
Since the excess biotin-phosphine elutes at a particular
time point as a distinct peak at the end of the gradient
(Figure 4c), its possible interference would be limited to
its co-eluting peptides. However, no obvious hindrance
has been observed for MS analysis of cross-linked
peptides due to excess biotin-phosphine. Ultimately,
elimination of these impurities is desirable and can be
accomplished by incorporating an acid cleavage site in
the cross-linkers. This is currently under investigation.
Figure 4. Enrichment of cross-linked Ac-myelin peptides after its
mixing with a Lysozyme digest (1:1) using Staudinger ligation. LC
MS traces of peptide mixtures: (a) before (inter-linked and dead-
end modified Ac-myelin eluted at 11.6 and 11.2 min, respectively)
and (b) after (inter-linked and dead-end modified Ac-myelin
eluted at 20.1 and 26.4 min, respectively) Staudinger ligation; (c)
after affinity purification and TFA cleavage elution. X-linked
Ac-myelin (14.4 min) indicates the enriched inter-linked peptide.
*Cleaved biotin-phosphine products. The dead-end modified Ac-
myelin eluted at 16.8 min.
spectrum of an inter-linked Ac-myelin product ([M ϩ
5H]5ϩ 644.33) after enrichment, purification, and elution
is illustrated in Figure 5a, in which two sets of major
ions were observed. One set of the ions (i.e., [M ϩ 4H]4ϩ
670.85, 675.36, 678.36) were the fragment ions resulting
from the parent ion ([M ϩ 5H]5ϩ 644.33), with a loss of
538.2, 520.2, and 508.2 Da, respectively. Two of the
corresponding losses (i.e., MHϩ 520.24 and 508.24) and
another fragment ion with MHϩ 447.19 were observed
as the second set of ions (labeled as I, II, and III) in
Figure 5a. The fragment ion with MHϩ 538.2 was often
not detected due to its further fragmentation into its
water loss product ion (MHϩ 520.2). Similarly, in the
MS/MS spectrum of the inter-linked Ac-IR7 after en-
richment and purification (Figure 5b), two sets of frag-
ment ions were observed, including one set of ions with
MSn Analysis to Facilitate Identification of
Cross-Linked Peptides
To confirm the cross-linked products, MS/MS analyses
were first carried out. As an example, the MS/MS

J Am Soc Mass Spectrom 2010, 21, 1432–1445
ENRICHMENT OF CROSS-LINKED PEPTIDES 1439
Figure 5. MSn analyses of inter-linked Ac-myelin and Ac-IR7 after Staudinger ligation and affinity
purification/TFA cleavage. MS/MS spectra of (a) inter-linked Ac-myelin ([M ϩ 5H]5ϩ 644.33); and (b)
inter-linked Ac-IR7 ([M ϩ 3H]3ϩ 773.73) peptides. MS3 spectra of (c) a fragment ion ([M ϩ 4H]4ϩ
,
670.85) after a loss of 538.2 Da from the parent ion ([M ϩ 5H]5ϩ 644.34) observed in (a); and (d) a
fragment ion ([M ϩ 2H]2ϩ 891.83) after a loss of 538.2 Da from the parent ion ([M ϩ 3H]3ϩ 773.73)
observed in (b). Characteristic fragment ions in MS/MS spectra, i.e., 447.2, 508.2, and 520.2 were
detected due to fragmentation in the cross-linker region and their structures are displayed in
Supplemental Figure 2.
MHϩ 447.28, 508.23, and 520.18, and another set of
region and resultant structures of the fragmentation
ions are depicted in Supplemental Figure 3. Therefore,
these ions (MHϩ 447.2, 508.2, 520.2) are characteristic
fragmentation ions of cross-linked products after
Staudinger ligation and affinity purification/TFA cleav-
age, and can be used as diagnostic markers to facilitate
the identification of cross-linked peptides by MS/MS.
Due to the preferential bond cleavages at the linker
region, limited fragmentation on the peptide backbone
was observed in MS/MS spectra. To obtain detailed
sequence information, MS3 analyses of the parent ions
after their characteristic losses were further performed.
Figure 5c displays the MS3 spectrum of the fragment ion
([M ϩ 4H]4ϩ 670.85), which resulted from the parent
inter-linked Ac-myelin ([M ϩ 5H]5ϩ 644.33) after a loss
fragment ions with [M ϩ 2H]2ϩ 891.83, 900.54, 906.47,
corresponding to the parent ion ([M ϩ 3H]3ϩ 773.73)
with a loss of 538.2, 520.2, or 508.2 Da, respectively.
Similar fragmentation pattern was detected in both
dead-end modified Ac-myelin and Ac-IR7 peptides
(Supplemental Figure 2). Taken together, the results
suggest that this type of fragmentation is unique to the
cross-linked peptides, and detection of a group of the
fragment ions (i.e., MHϩ 447.2, 508.2, and 520.2) is
independent of the type and sequence of the cross-
linked peptides. This prompted us to believe that such
fragmentation is due to unique bond cleavages at the
conjugated cross-linker region during CID analysis. The
proposed cleavage sites in the conjugated cross-linker

1440 VELLUCCI ET AL.
J Am Soc Mass Spectrom 2010, 21, 1432–1445
of 538.2 Da as measured in Figure 5a. Based on the
previously described terminology of inter-linked pep-
tides and their fragment ions [8], we have labeled one
sequence of the inter-linked Ac-myelin as the ␣ chain
and the other as the ␤ chain. In addition to fragment
ions belonging to ␣ or ␤ chain sequence, fragment ions
containing both chains were detected. The masses of all
possible fragment ions of the inter-linked Ac-myelin
were calculated, which were then manually matched to
the observed ions in Figure 5c for unambiguous iden-
tification of the inter-linked Ac-myelin sequence. Simi-
larly, MS3 analysis of the fragment ion ([M ϩ 2H]2ϩ
891.83) of the parent inter-linked Ac-IR7 ion ([M ϩ
3H]3ϩ 773.73) after a loss of 538.2 Da (Figure 5b)
confirmed its sequence as illustrated in Figure 5d.
mass modification would be undefined since it contains
both the cross-link (i.e., 631.24 Da) and an additional
covalently attached peptide. Even with mass modifica-
tion search, most of the cross-linked peptides were not
identified by Batch-tag search. This is a result of the
complicated fragmentation in MS/MS spectra caused
by preferred cleavage at the conjugated linker region
and the presence of an additional sequence in the
inter-linked peptides.
To determine the cross-linked peptide identities,
three additional steps were carried out. First, the mo-
noisotopic masses and charges of all parent ions se-
lected for MS/MS were subjected to the MS-bridge
program to identify putative azide-DSG cross-linked
peptides of cytochrome c by peptide mass mapping
with high mass accuracy (Ͻ20 ppm) [62]. Second, we
manually examined MS/MS spectra of the putative
cross-linked cytochrome c peptides identified in MS-
bridge to determine whether the diagnostic fragment
ions (MHϩ, 447.2, 508.2, 520.2) of the conjugated cross-
link were observed. Third, MS-tag, MS-product and
manual inspection were performed to identify and
confirm the cross-linked peptide sequences and corre-
late them with MS-bridge search results. For instance,
based on MS-bridge search results, an ion with [M ϩ
4H]4ϩ 542.54 matched to an inter-linked peptide [Ac-
GDVEKGK inter-linked to KIFVQK], an ion with [M ϩ
3H]3ϩ 526.96 matched to an intra-linked peptide
[GK(631.24)KIFVQK], and an ion with [M ϩ 3H]3ϩ
745.34 matched to a dead-end product [K(649.26)
TGQAPGFSYTDANK]. Consistent with cross-linked
model peptides (Figure 5a and b, and Supplemental
Figure 2), all of the putative cross-linked cytochrome c
peptides gave same characteristic fragment ions (MHϩ
447.2, 508.2, and 520.2) in their MS/MS spectra as
illustrated in Figure 6a–c, suggesting that these pep-
tides are indeed enriched cross-linked products. Addi-
tionally, the corresponding fragment ions of parent ions
with a loss of 508.2 or 520.2 Da were observed in their
MS/MS spectra (Figure 6a–c).
Enrichment and Identification of Cytochrome c
Cross-Linked Peptides
To determine whether the cross-linking enrichment
strategy can be applied to more complex samples,
cytochrome c was used as a model protein since it has
been widely used in other cross-linking experiments [3,
8, 40, 41, 65–68]. Cytochrome c was cross-linked using
azide-DSG and the cross-linked products were sepa-
rated by 1-D SDS-PAGE. Various molar ratios of azide-
DSG and cytochrome c (10:1, 15:1; 20:1, 50:1) were tested
to optimize the cross-linking efficiency with tenfold
excess of azide-DSG determined as the optimal ratio for
final analyses. After cross-linking, the cross-linked cy-
tochrome c was digested in-solution with trypsin and
the tryptic peptides were subjected to Staudinger liga-
tion using the biotin-phosphine (S-17) as described
above (Figures 1 and 2). The resulting conjugates were
then affinity purified, eluted with TFA, and analyzed by
LC MS/MS. In comparison with model peptides, data
processing of cross-linked cytochrome c peptides was
much more complicated due to the presence of a large
number of heterogeneous and unknown cross-linked
peptides. To facilitate the data interpretation, we have
employed a suite of software (i.e., Batch-tag, MS-bridge,
MS-tag, MS-product and Search Compare) in Protein
Prospector to identify cytochrome c peptides via a
method similar to that previously described [62].
MS/MS spectra were first searched against cytochrome
c sequence using Batch-tag with mass modification
search to identify unmodified peptides as well as pep-
tides with various mass modifications. Our cross-linker
targets lysine residues and the free protein N-terminus,
therefore we considered these residues as potential
sites for mass modifications [62]. For dead-end (type 0)
modified and intra-linked (type 1) peptides, defined
mass modifications on lysine residues, i.e., 649.26 and
631.24 Da, would be observed, respectively. These
masses are attributed to the addition of cross-links on
the peptides due to the cross-linking nature of the type
0 and 1 products [8], which can be used for their
identification. However, for inter-linked peptides, the
In addition to MS/MS experiments, MS3 analyses of
selected fragment ions observed in MS/MS spectra of
these three cross-linked cytochrome c peptides (Figure
6a–c) were carried out to further confirm their identi-
ties. For the inter-linked peptide ([M ϩ 4H]4ϩ 542.54),
its fragment ion with [M ϩ 2H]2ϩ 824.72 (Figure 6a) was
selected for MS3 analysis. The loss of 520.2 Da from the
parent ion ([M ϩ 4H]4ϩ 542.54) led to a mass modifica-
tion of 112 Da on the inter-linked fragment ion ([M ϩ
2H]2ϩ 824.72) due to the remnant of the conjugated link
(see Supplemental Figure 3). As shown in Figure 6d, a
series of b and y ions for either a single or both peptide
chains were observed in the MS3 spectrum of [M ϩ
2H]2ϩ 824.72, further confirming the inter-linked pep-
tide sequences identified by MS-bridge. For the intra-
linked peptide ([M ϩ 3H]3ϩ 526.96), the fragment ion
with [M ϩ 2H]2ϩ 536.31 (Figure 6b) was analyzed by
MS3, which has a mass modification of 124 Da due to
the loss of 508.2 Da from its intra-linked parent ion. In

J Am Soc Mass Spectrom 2010, 21, 1432–1445
ENRICHMENT OF CROSS-LINKED PEPTIDES 1441
Figure 6. MSn analyses of three azide-DSG cross-linked peptides of cytochrome c after Staudinger
ligation and affinity purification/TFA cleavage. MS/MS spectra of the selected (a) inter-linked ([M ϩ
4H]4ϩ 542.54); (b) intra-linked ([M ϩ 3H]3ϩ 526.96); and (c) dead-end ([M ϩ 3H]3ϩ 745.35) modified
peptides. Characteristic fragment ions (MHϩ 447.2, 508.2, 520.2) were detected in all spectra. MS3
spectra of (d) a fragment ion ([M ϩ 2H]2ϩ 824.72) observed in (a), after a loss of 508.2 Da from the
parent ion ([M ϩ 4H]4ϩ 542.54); (e) a fragment ion ([M ϩ 2H]2ϩ 536.31) observed in (b), after a loss
of 508.2 Da from the parent ion ([M ϩ 3H]3ϩ 526.96); and (f) a fragment ion ([M ϩ 2H]2ϩ 863.80)
observed in (c), after a loss of 508.2 Da from the parent ion ([M ϩ 3H]3ϩ 745.35). The identified
cross-linked peptide sequences are displayed in (d)-(f). dn ϭ dead-end modification.
the MS3 spectrum of [M ϩ 2H]2ϩ 536.31 (Figure 6e),
the dead-end modified peptide with a sequence of
K(dn)TGQAPGFSYTDANK, in which the N-terminal
lysine was modified.
In this work, although all of the cross-linked pep-
tides have given characteristic losses in MS/MS for
their identification, we were only able to interpret four
additional MS3 spectra, including two inter-linked and
two intra-linked cytochrome c peptides as displayed in
Supplemental Figures 4 and 5. This is due to lower
sensitivity in MS3 and lack of software for effective
detection of y2-y5 and b3-b7 ion series allowed unam-
biguous identification of the intra-linked peptide as
GKKIFVQK, in which the two adjacent lysine residues
at the N-terminus were determined to be cross-linked.
Similar fragmentation was also observed in the MS3
spectrum (Figure 6f) of a MS/MS fragment ion with
[M ϩ 2H]2ϩ 863.80 (Figure 6c) from the dead-end
modified parent ion ([M ϩ 3H]3ϩ 745.35) after a loss of
508.2 Da. As shown, y2-y14 and b2-b14 ions confirmed

1442 VELLUCCI ET AL.
J Am Soc Mass Spectrom 2010, 21, 1432–1445
Table 1. Summary of identified cross-linked peptides of cytochrome c after enrichment
m/z
Seen before
Type
Peptide sequence
Ac-GDVEKGK
Ac-GDVEKGKK
KIFVQK
GGKHK
KTGQAPGFSYTDANK
AA location
(Observed)
z
⌬(ppm)
enrichment
References
0
0
0
0
0
G1-K7
G1-K8
712.3342
517.9262
706.3799
588.2895
745.3507*
559.2618
826.0621
619.7963
728.3737
485.9183
569.7707
871.4472
581.3033
606.2740
767.3693
717.0482
789.9327
526.9579*
395.4689
769.7238
577.5500*
462.2401
609.5687
487.8605
406.7162
1229.5850
820.0587
615.2941
833.9532
556.2999
841.9451
561.6340*
624.8149
416.8803
827.7458
621.0619
1070.0338
713.6908
723.0504
542.5392*
560.6229
420.7197
506.2615
2
3
2
2
3
4
3
4
2
3
2
2
3
2
2
3
2
3
4
3
4
5
4
5
6
2
3
4
2
3
2
3
2
3
3
4
2
3
3
4
3
4
4
5
9
7
6
7
2
5
1
5
5
1
1
5
2
Ϫ5
7
6
5
3
Ϫ1
8
5
Ϫ1
7
3
6
5
2
10
1
3
5
4
6
5
6
7
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
K8-K13
G23-K27
K39-K53
[40,41,67]
[40,41,66,67]
[40,65,66,67]
[41,67]
0
0
KTGQAPGFSYTDANKNK
KYIPGTK
K39-K55
K73-K79
0
0
KGER
EDLIAYLKK
K88-R91
E92-K100
[67]
0
1
1
1
KATNE
K100-E104
G1-K8
G1-K13
G6-K13
Ac-GDVEKGKK
Ac-GDVEKGKKIFVQK
GKKIFVQK
[41,66,67]
1
1
1
GGKHKTGPNLHGLFGR
GGKHKTGPNLHGLFGRK
KTGQAPGFSYTDANKNK
G23-R38
G23-K39
K39-K55
[8,40,41,66,67]
[66,67]
[8,40,41,66,68]
[8,40,41,66,67]
1
1
1
1
1
2
2
2
2
2
2
2
MIFAGIKKK
M80-K88
MoxIFAGIKKK
KKGER
M80-K88
K87-R91
[41]
GEREDLIAYLKKATNE
EDLIAYLKKATNE
G89-E104
E92-E104
[67]
6
6
5
6
7
7
Ac-GDVEKGK
KIFVQK
Ac-GDVEKGK
KK
Ac-GDVEKGK
KKGER
Ac-GDVEKGKK
KK
Ac-GDVEKGKK
KKGER
GKK
KGER
GKK
G1-K7 ϳ K8-K13
G1-K7 ϳ K87-K88
G1-K7 ϳ K87-R91
G1-K8 ϳ K87-K88
G1-K8 ϳ K87-R91
G6-K8 ϳ K88-R91
G6-K8 ϳ K100-E104
[3,40,41,67]
[67,68]
[67,68]
[67]
452.7451
538.2844
726.3632
4
4
2
10
5
Yes
Yes
Yes
[67,68]
Ϫ17
762.8826*
508.9219
381.9450
652.3468
489.5106
573.6183*
2
3
4
3
4
3
6
1
5
9
6
6
Yes
Yes
Yes
Yes
Yes
Yes
[66,68]
KATNE
2
2
2
2
KIFVQK
KATNE
GGKHK
KATNE
KTGQAPGFSYTDANK
MIFAGIKKK
KTGQAPGFSYTDANK
KATNE
K8-K13 ϳ K100-E104
K23-K27 ϳ K100-E104
K39-K53 ϳ M80-K88
K39-K53 ϳ K100-E104
813.4261
650.9429
695.0784
4
5
4
13
13
2
No
No
Yes
[67,68]

J Am Soc Mass Spectrom 2010, 21, 1432–1445
ENRICHMENT OF CROSS-LINKED PEPTIDES 1443
Table 1. Continued
m/z
Seen before
Type
2
Peptide sequence
KKGER
KATNE
KGER
KATNE
KATNE
KATNE
AA location
(Observed)
z
⌬(ppm)
enrichment
References
K87-R91 ϳ K100-E104
453.2339
561.2752
585.6121
4
11
Yes
2
2
K88-R91 ϳ K100-E104
3
3
7
Yes
Yes
K100-E104 ϳ K100-E104
10
All of the cross-linked peptides have displayed characteristic losses in MS/MS.
*These cross-linked peptides have been further confirmed by MS3.
searching of MS3 spectra with complex fragmentation,
which result from cross-linked peptides. Further soft-
ware development that allows automated interpreta-
tion of MS3 spectra of enriched cross-linked peptides
will significantly facilitate the identification process by
peptide sequencing and increase the confidence of
sequence assignments.
sines), 13 linkages have the distances between their ␣
carbons within 20 Å, and 3 of them have the distances
ϳ21–25 Å, which was measured using Jmol (http://
jmol.org/). This is consistent not only with the length of
a fully expanded azide-DSG (7.7 Å spacer length) and
two lysine side chains, but also with the previous
results using similar lengths of NHS ester cross-linkers
[39, 67, 70]. The results suggest that our cross-linking
condition did not induce significant disturbance to
cytochrome c structural conformations.
As summarized in Table 1, 14 dead-end modified
(type 0), 24 intra-linked (type 1), and 20 inter-linked
(type 2) peptides of cytochrome c have been identified
from our new enrichment strategy using Staudinger
ligation. This represents 35 unique cytochrome c cross-
linked sequences, among which 19 have been previ-
ously reported using other NHS ester cross-linking
reagents [3, 8, 40, 41, 65–68], and 16 were first identified
in this study. To have a better comparison, we have
summarized a combined non-redundant list of inter-
linked, intra-linked, and dead-end modified lysine res-
idues of bovine cytochrome c identified in this work
and other reports [3, 8, 65, 66, 68] (Supplemental Table
1). As shown, a total of 19 putative inter-linked, 8
intra-linked, and 10 dead-end modified lysine residues
have been identified by MS analyses using various
cross-linkers. Among them, our study has identified 12
inter-linked lysines including eight new ones, eight
intra-linked lysines including four new ones, and 10
dead-end modified lysines including nine new ones.
The absence of a few previously reported linkages may
be due to the facts that we have only listed out the
cross-linked peptides with characteristic diagnostic ions
in Table 1, which do not include peptides identified
based solely on the m/z and charge state as other papers
had in the past. In addition, different cross-linkers with
various spacer lengths and experimental conditions
may result in identification of different peptides.
In comparison with the results before enrichment,
the Staudinger ligation-based enrichment strategy has
demonstrated its effectiveness in isolating cross-linked
products. Based on the three-dimensional crystal struc-
ture of bovine heart cytochrome c (PDB ID; 2B4Z) [69],
Conclusion
In this study, we have designed, synthesized, and
evaluated a novel azide-tagged cross-linker, azide-DSG,
which enables selective and effective enrichment of
cross-linked peptides using Staudinger ligation. In com-
parison with other types of enrichment strategies using
alkyne tags [39], the azide tag is more resistant to oxida-
tive side reactions that could reduce the capture efficiency.
Although azide-alkyne chemistry has been applied to
capture azide-tagged cross-linked peptides [40, 41], we
demonstrate that the new Staudinger ligation-based
strategy is efficient for selective enrichment of the
cross-linked peptides from complex mixtures, and is
better suited for peptide analysis. In addition, the
diagnostic ions (MHϩ 447.2, 508.2, 520.2) detected in
MS/MS spectra due to characteristic fragmentation in
the conjugated link after Staudinger reaction and affin-
ity purification offer a unique means for MS identifica-
tion with increased confidence. This set of diagnostic
ions is easy to detect and would be more reliable for
identifying cross-linked peptides by MS in comparison
with a diagnostic fragmentation with a neutral loss as
employed in alkyne tagged cross-linked peptides [39].
Moreover, MS3
analysis permitted additional fragmen-
tation information for unambiguous peptide identifica-
tion. In comparison with immobilized cyclic alkyne
[41], synthesis of biotin-phosphine is much simpler
with only three steps. Taken together, the novel enrich-
ment strategy developed in this work helps facilitate
MS identification of cross-linked peptides from com-
plex mixtures. With the development of bioinformatics
tools, we anticipate that this approach can be easily
generalized for capturing and identifying cross-linked
peptides of protein complexes.
we have calculated the
␣ carbon distances between the
cross-linked lysine residues identified in the cross-
linked peptides. As shown in Supplemental Table 2,
among 16 different combinations of type 1 or 2 cross-
linked lysines in cytochrome c identified in this work
(not considering linkages between two adjacent ly-

1444 VELLUCCI ET AL.
J Am Soc Mass Spectrom 2010, 21, 1432–1445
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Acknowledgments
The authors thank Drs. Christian Tagwerker and Lei Fang for
initial analysis and members of the Huang and Rychnovsky
laboratory for their help during this study. They thank Professor
A.L. Burlingame, Peter Baker, and Aenoch Lynn at UCSF for using
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work by National Institutes of Health grants (GM-74,830 to L.H.).
Appendix A
Supplementary Material
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