Novel Concepts of MS-Cleavable Cross-linkers for Improved Peptide Structure Analysis

  • Christoph Hage
  • Francesco Falvo
  • Mathias Schäfer
  • Andrea Sinz
Research Article


The chemical cross-linking/mass spectrometry (MS) approach is gaining increasing importance as an alternative method for studying protein conformation and for deciphering protein interaction networks. This study is part of our ongoing efforts to develop innovative cross-linking principles for a facile and efficient assignment of cross-linked products. We evaluate two homobifunctional, amine-reactive, and MS-cleavable cross-linkers regarding their potential for automated analysis of cross-linked products. We introduce the bromine phenylurea (BrPU) linker that possesses a unique structure yielding a distinctive fragmentation pattern on collisional activation. Moreover, BrPU delivers the characteristic bromine isotope pattern and mass defect for all cross-linker-decorated fragments. We compare the fragmentation behavior of the BrPU linker with that of our previously described MS-cleavable TEMPO-Bz linker (which consists of a 2,2,6,6-tetramethylpiperidine-1-oxy moiety connected to a benzyl group) that was developed to perform free-radical-initiated peptide sequencing. Comparative collisional activation experiments (collision-induced dissociation and higher-energy collision-induced dissociation) with both cross-linkers were conducted in negative electrospray ionization mode with an Orbitrap Fusion mass spectrometer using five model peptides. As hypothesized in a previous study, the presence of a cross-linked N-terminal aspartic acid residue seems to be the prerequisite for the loss of an intact peptide from the cross-linked products. As the BrPU linker combines a characteristic mass shift with an isotope signature, it presents a more favorable combination for automated assignment of cross-linked products compared with the TEMPO-Bz linker.

Key words

Bromine isotope pattern Chemical cross-linking Free-radical-initiated peptide sequencing Tandem mass spectrometry TEMPO-Bz linker 





Amine fragment of bromine phenylurea cross-linker


Isocyanate fragment of bromine phenylurea cross-linker


Bromine phenylurea




Collision-induced dissociation


Dimethyl sulfoxide


Electron capture dissociation


Electrospray ionization


Electron transfer dissociation


Free-radical-initiated peptide sequencing


Higher-energy collision-induced dissociation


Mass spectrometry


Normalized collision energy






Triethylamine acetate




Chemical cross-linking in combination with mass spectrometry (MS) has emerged as a promising technique for studying protein conformation as well as protein–protein and protein–ligand interactions [1, 2, 3, 4, 5, 6]. Because of the great variety of commercially available cross-linking reagents and standardized protocols, the cross-linking/MS approach has become a powerful tool for studying in vitro and in vivo protein systems [7, 8, 9]. The major choices to be made comprise the selection of suitable reactive groups that determine cross-linker reactivity (homobifunctional or heterobifunctional) and the proper spacer length of the linker. Cross-linkers should be sufficiently long (spacer length approximately 15 Å) to allow a reaction between protein interaction partners; on the other hand they should be short (approximately 5 Å) to allow a computational modeling based on the spatial distance constraints imposed by the cross-links [10, 11, 12, 13, 14]. A liquid-chromatography-based reversed-phase separation of cross-linked peptides preceding the MS analysis can be regarded as a standard element in sample preparation. In addition, further prefractionation steps (e.g., by size exclusion or strong cation exchange chromatography) are beneficial for enrichment of cross-linked products [15, 16, 17]. However, improved enrichment and separation tools do not necessarily guarantee better cross-link identification. Various attempts have been made to allow unambiguous MS identification of the low-abundance cross-linked products frequently produced, ideally requiring the complete sequencing of both cross-linked partners. One of the drawbacks in chemical cross-linking combined with MS is that in many cases only one of the connected peptides is thoroughly sequenced, and there are no fragmentation data available for the other peptide [18, 19]. Since different combinations of cross-linked peptides might yield isobaric species, it is desirable to determine the intact masses of the cross-linked peptides and thoroughly sequence both peptides.

To efficiently handle the challenges involved in the identification of cross-linked products, three general attempts have been made: First, incorporation of stable isotopes (2H or 13C) in the spacer of the cross-linker allows the identification of cross-links by a directed search for characteristic isotopic patterns as usually isotope-coded and noncoded cross-linking reagents are used in a 1:1 ratio [20]. Isotope doublets with distinct mass differences are identified not only at the MS level but also at the MS/MS level, and will therefore serve as proof of a “true” cross-link [21, 22, 23, 24]. Fragmentation efficiencies are not changed by this approach, and the fundamental problem of varying fragmentation efficiencies of the cross-linked peptides remains unsolved. The power of the isotope-labeling approach is further increased by use of mass defects of heteroatoms [25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35]. A characteristic mass-deficient modification in combination with a unique isotopic pattern is obtained when bromine-containing labeling reagents are used. Bromine exhibits a natural isotope distribution with two nuclides, separated by 1.998 u, with nearly equal intensities originating from 50.69% 79Br (78.9183 u) and 49.31% 81Br (80.9163 u). This “bromine isotope signature” has been used for peptide sequencing [36, 37, 38] and for localizing phosphosites [39]. Not surprisingly, the high mass defect of bromine (-0.82 u) has proven especially useful for the MS-based identification of labeled peptides [28, 31, 32, 33, 35] as it is more pronounced than the mass defect resulting from phosphorylation (-0.70 u) [25, 40, 41].

Second, specific fragmentation of the cross-linker will improve the identification of cross-linked products. The use of different fragmentation techniques, such as collision-induced dissociation (CID) in a low collision and activation energy regime [42], higher energy CID (HCD), and electron transfer dissociation (ETD)/electron capture dissociation (ECD) [43, 44], may result in complementary fragmentation patterns for a facilitated cross-link identification [45, 46, 47]. Collision-based methods are the state of the art in proteomics applications, with resonant activation in ion traps being the most commonly used fragmentation technique. In contrast to the collisional activation methods, ETD and ECD yield c- and z-type ions, but the reduction of the precursor ion is frequently observed [48, 49]. Also, duty cycles are limited by the reaction time of the ETD reagent. Relatively low fragment ion yields, the need for high precursor charge states, and the time-consuming ETD workflow pose limitations for conducting parallel or sequential CID/ETD fragmentation experiments for all precursor ions [47, 50, 51].

One novel concept to overcome the limitations of sequential fragmentation experiments with CID and ETD is to perform free-radical-initiated peptide sequencing (FRIPS) [52, 53, 54]. FRIPS relies on a CID-labile reagent forming open-shell peptide product ions on collisional activation. When the cross-linked peptides are analyzed in negative electrospray ionization (ESI) mode, all competing fragmentation pathways are suppressed and homolytic bond cleavage of the cross-linker leads to the primary formation of the desired peptide radical product ions. These initially formed product ions undergo further radical-driven fragmentations, generating additional ETD-like fragments that allow sequencing of the connected peptides and pinpointing of the cross-linking sites [55, 56].

Finally, specifically designed MS-cleavable cross-linkers further facilitate the unambiguous identification of cross-linked products [57]. Here, the spacer arm of the MS-cleavable cross-linker is designed to contain a CID-cleavable bond, such as the labile Asp–Pro peptide bond [58, 59, 60], a (thio)urea group [61, 62, 63], or a sulfoxide [64, 65, 66]. Alternatively, cross-linkers with ETD-cleavable disulfides or hydrazones [67, 68] and photocleavable cross-linkers have been developed [7, 69, 70]. The reproducible fragmentation of labile groups in the cross-linker chain results in the formation of two specifically decorated peptide fragment ions. Hence, the characteristically mass-shifted peptide product ions are unambiguously determined on the basis of the predictable and unique fragmentation pattern of the cross-linker [47].

Since the design of MS-cleavable cross-linkers does not preclude the additional use of isotope coding, we aimed to test the combination of complementary strategies to further improve the sensitive and effective identification of peptide cross-links. In the two cross-linking principles presented in this article, we rely on reagents combining MS cleavability with bromine isotope coding, as in the novel bromine phenylurea (BrPU) linker, or FRIPS, as in the recently introduced TEMPO-Bz linker, which consists of a 2,2,6,6-tetramethylpiperidine-1-oxy (TEMPO) moiety connected to a benzyl (Bz) group. To test this multidimensional approach, we compare the fragmentation behavior of five cross-linked peptides in negative ESI experiments on collisional activation (CID and HCD) performed with an Orbitrap Fusion mass spectrometer. As we had previously observed the loss of intact peptide chains from cross-linked peptides containing acidic residues when using the TEMPO-Bz linker in negative ESI experiments, we also sought to further elucidate this characteristic fragmentation behavior [56].


Chemicals and Peptides

All chemicals and solvents were used without further purification (Acros Organics, Aldrich, Fluka, Merck). Human angiotensin II was purchased from Sigma-Aldrich, and test peptide 1 was obtained from Creative Molecules. The tripeptides EAA, DAA, and VVA were kindly provided by Mike Schutkowski (Martin Luther University Halle-Wittenberg).

Synthesis of Cross-linkers

Synthesis of the BrPU linker is described in the electronic supplementary material; synthesis of the TEMPO-Bz linker was performed as described previously [71].

Cross-linking Experiments

Experiments with the Tripeptides EAA, DAA, and VVA

Three microliters of triethylamine (TEA) was added to one peptide aliquot [2 μl, 20 mM in dimethyl sulfoxide (DMSO)] to give 5 μl of 8 mM peptide solution. One equivalent of cross-linker (5 μl, 8 mM in DMSO) was added, and after 30 min at room temperature, 1 μl water was added to hydrolyze unreacted cross-linker overnight. Samples were then diluted with 0.18 M triethylamine acetate (TEAAc) solution to give a final volume of 133 μl (300 μM peptide solution containing approximately 5% DMSO). Samples were loaded on C18 ZipTip columns (Millipore) that had been equilibrated with acetonitrile (ACN) and 0.1M TEAAc, washed five times with 0.1M TEAAc, and stored at 4 °C. Before MS experiments, the samples were washed four times with 10% (v/v) ACN; for VVA, 30% (v/v) ACN. Elution was performed three times with 5-μl aliquots of 0.8% (v/v) TEA in 80% (v/v) methanol. Aliquots were combined to yield a final sample volume of 15 μl.

Experiments with Angiotensin II and Test Peptide 1

DMSO (3.5 μl), TEAAc solution (0.5 μl), and TEA (0.5 μl) were added to one peptide aliquot (2 μl, 10 mM in H2O). To this solution, 3.5 μl of 40 mM cross-linker solution (in DMSO) was added to give 10 μl of a 2 mM peptide solution in 70% (v/v) DMSO. The reaction was allowed to proceed overnight at room temperature. Then, 40 μl TEA (80 mM)–TEAAc (20 mM) solution was added to give a 400 μM peptide solution [14% (v/v) DMSO]. Samples were loaded on C18 ZipTip columns and washed seven times with 0.1 M TEA–TEAAc. Samples were eluted as described earlier, but washed with 40% (v/v) ACN before elution. Test peptide 1 was cross-linked and digested with trypsin according to a previous protocol [56].

Offline Nano-ESI Orbitrap MS

Samples (3 μl) were applied to self-made capillaries, and MS analysis was performed with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) with a nano-ESI source (Nanospray Flex, Thermo Fisher Scientific). Capillaries (4-in., 1.2-mm outer diameter, 0.68-mm inner diameter, fire-polished borosilicate glass capillaries with filament, World Precision Instruments) were pulled in-house (model P-1000 Flaming/Brown-style micropipette puller, Sutter Instrument) and gold coated. The capillary voltage was set between -1.0 and -1.2 kV and the source temperature was held at 275 °C. MS data were collected in the m/z range from 150 to 2000. For MS/MS measurements, ions were isolated in the quadrupole with an isolation window of 2 Th (TEMPO-Bz linker) and 4 Th (BrPU linker), fragmented by CID and HCD (0–30% normalized collision energy; NCE), and analyzed in the Orbitrap (R = 60,000 at m/z 200) with external calibration (experimental error less than 5 ppm) or internal calibration (experimental error less than 3 ppm). To avoid adduct formation of cross-linked peptides, in-source activation was applied (30–50 eV).

Results and Discussion

Nomenclature of Fragment Ions

BrPU Linker

We have synthesized a homobifunctional amine-reactive, N-hydroxysuccinimide (NHS) ester cross-linker containing two amine bromobenzoic acid moieties connected by a central urea group (see the electronic supplementary material). We termed our novel cross-linker “bromine phenylurea” (BrPU): bis(2,5-dioxopyrrolidine-1-yl) 4,4′-(carbonylbis(azanediyl))bis(3-bromobenzoate). CID of the urea group proceeds in a nonsymmetrical fashion giving rise to an amine (BrAm) and an isocyanate (BrIc; Fig. 1). The unique structure of BrPU leads to the previously described urea-typical fragmentation pattern exhibiting two doublets with a distinct mass difference of 25.979 u [61], and additionally imprints the bromine isotope pattern for all cross-linker-decorated fragments (Figs. S1, S2).
Fig. 1

Reactivity of the BrPU linker with amine groups in peptides and nomenclature of the fragments created in created in negative electrospray ionization (ESI)–collision-induced dissociation (CID) tandem mass spectrometry (MS/MS)

TEMPO-Bz Linker

The previously described homobifunctional amine-reactive TEMPO-Bz linker [56, 71] is CID-cleavable {(1-[2-(2,5-dioxopyrrolidine-1-yloxycarbonyl)benzyloxy]-2,2,6,6-tetramethylpiperidine-4-carboxylic acid 2,5-dioxopyrrolidine-1-yl ester} and contains a TEMPO moiety connected to a Bz group. CID of the central NO–C bond proceeds exclusively in a homolytic fashion during collisional activation in negative ESI experiments, resulting in a benzoyl radical (Bz•) and a TEMPO• radical (Fig. 2; see Fig. S3 for a comparison of positive and negative ESI-MS). In negative ESI mode, product ion CID experiments trigger a homolytic cleavage that results in the exclusive formation of a characteristic doublet with a distinct mass difference of 65.084 u. Whereas the stable TEMPO• radical undergoes a limited number of further radical-driven fragmentations, the more reactive Bz• radical initiates the migration of the radical site within the fragment ion, leading to various radical-driven fragmentations (Fig. S4). These give rise to c- and z- type ions, in addition to a- and x-type ions, and neutral losses from amino acid side chains (Fig. S5) [56].
Fig. 2

Reactivity of the TEMPO-Bz linker with amine groups in peptides and nomenclature of the fragments created in negative ESI–CID-MS/MS

Choice of Model Peptides for Cross-linking

We selected three tripeptides as simple model systems for cross-linking, encompassing the sequences EAA, DAA, and VVA, as they contain only the N-terminus as a reaction site with the NHS ester linkers used here. The sequences of these peptides were designed on the basis of our previous hypothesis on the presence of an N-terminal acidic amino acid that triggers the loss of an intact peptide from the cross-linked product [56]. On cross-linking, covalently connected peptide dimers are obtained. As additional model peptides, we selected angiotensin II (DRVYIHPF) and test peptide 1 with an acetylated N-terminus (Ac-TRTESTDIKRASSREADYLINKER), which have already been used by us in cross-linking/FRIPS studies [56].

BrPU Cross-linking

For all three cross-linked tripeptides (EAA, DAA, and VVA), HCD yields a higher number of fragment ions than CID (Figs. 3a–c, S6a–c) [72]. The negative ESI–CID spectra, recorded at 15% NCE, show the precursor ions of the cross-linked peptide dimers as doubly negatively charged signals, with only minor amounts (relative intensities of 1–8%) of cross-linker fragment ions. On the other hand, HCD spectra recorded at the identical NCE also revealed linker fragments, partially with neutral losses (water, ammonia, CO2), as well as peptide fragments. The different fragment ions created in CID versus HCD originate from the nature of collisional activation—namely, resonant activation in the ion trap versus beam-type activation—causing a higher number of secondary fragmentation events in HCD. For all three peptides, the characteristic BrIc and BrAm fragments of the cross-linker were observed. The loss of a complete peptide was the base peak for the cross-linked DAA dimer, according to the signal at m/z 239.066 (Fig. 3b), confirming our hypothesis of an N-terminal acidic amino acid to be the requirement for this fragmentation. The loss of an intact peptide was not observed for the cross-linked EAA dimer, underlining the importance of an N-terminal aspartic acid residue.
Fig. 3

CID (upper panel) and higher-energy CID (HCD; lower panel) fragment ion mass spectra for peptides cross-linked with the BrPU linker: a EAA, the bromine isotope pattern is shown as an inset; b DAA; c VVA; d angiotensin II; e test peptide 1. Spectra were recorded with 15% normalized collision energy (NCE) (EAA, DAA, and VVA) and 20% NCE (angiotensin II and test peptide 1). Gua guanidine group. Asterisk signal is not assigned

For BrPU-cross-linked angiotensin II, the differences between CID and HCD fragment ion spectra (0–30% NCE) are not as pronounced as for the tripeptides (Figs. 3d, S6d). The most prominent signals in the fragment ion mass spectra recorded at 20% NCE correspond to the BrIc and BrAm fragments of the BrPU linker. Other high-abundance signals represent the loss of a peptide from the cross-linked angiotensin II dimer, which is attributed to the presence of an N-terminal aspartic acid. Angiotensin II contains a second potential reaction site for NHS esters at Tyr-4 in addition to the N-terminus; however, it was never observed to participate in the cross-linking reactions. The presence of the y7 ion in the fragment ion mass spectra indicates an exclusive reaction of the N-terminus with BrPU.

In contrast to the other peptides used, test peptide 1 was tryptically digested after the cross-linking reaction, resulting in the fragments TESTDIKR and EADYLINKER, which are linked via their lysine residues. In CID fragment ion spectra recorded at 20% NCE, the linker fragments BrIc and BrAm are almost exclusively observed as singly and doubly negatively charged species (Fig. 3e). In HCD, peptide backbone fragmentation is visible together with a prominent linker fragmentation, including additional neutral losses, but we never observed the loss of a complete peptide as exclusively internal cross-links are created. In general, HCD yielded richer spectra than CID for test peptide 1 (Fig. S6e).

TEMPO-Bz Cross-linking

Cross-linking studies of angiotensin II and test peptide 1 have been already performed by us with the TEMPO-Bz linker using CID at 25% NCE [56, 71]. Here, we compare negative ESI–CID and negative ESI–HCD conditions for the three tripeptides EAA, DAA, and VVA as well as for angiotensin II and test peptide 1. In particular, we aimed to investigate more closely the loss of a complete peptide from the cross-linked product that we had observed in our previous studies. For EAA, DAA, and VVA, the CID fragment ion mass spectra (15% NCE) are almost identical, showing the precursor ion of cross-linked peptide dimers as base peaks, with only negligible amounts of fragment ions originating from the linker (Figs. 4a–c, S7a–c). The HCD spectra (15% NCE) exhibit a completely different behavior: For EAA and VVA, the TEMPO•-modified peptide presents the most abundant species (Fig. 4a, c), whereas for DAA, the intact cross-linked peptide dimer is the base peak (Fig. 4b). Also, peptide backbone fragments as well as neutral losses are observed. As for the BrPU linker, the loss of an intact peptide chain is found only for DAA, but not for EAA and VVA. As aspartic acid is able to form a six-membered intermediate, it is prone to induce the fragmentation of an adjacent amide bond in the peptide. In the case of an N-terminal aspartic acid, there are two neighboring amide bonds (the newly created amide bond to connect the peptide with the cross-linker and the peptide bond connecting the first and second amino acids), which can be cleaved. As glutamic acid possesses one additional methylene group in its side chain compared with aspartic acid, it does not induce this specific fragmentation. Further elucidation of the exact mechanism of this reaction will be the topic of future studies.
Fig. 4

CID (upper panel) and HCD (lower panel) fragment ion mass spectra for peptides cross-linked with the TEMPO-Bz linker: a EAA; b DAA; c VVA; d angiotensin II; e test peptide 1. Spectra were recorded with 15% NCE (EAA, DAA, and VVA) and 20% NCE (angiotensin II and test peptide 1). Asterisk signal is not assigned

CID at 20% NCE of TEMPO-Bz-cross-linked angiotensin II showed the doubly negatively charged cross-linked dimer at m/z 1194.103 as the most prominent species. Also, a number of low-abundance signals are visible for linker fragments, partially with neutral losses (water, ammonia, CO2) or the loss of a methyl radical (Fig. 4d). As for the three tripeptides, HCD produced much richer spectra, showing extensive peptide backbone fragmentation (a-, x-, and z-type ions) as well as fragmentation of the linker and the loss of an intact peptide chain (m/z 1044.528) (Fig. S7d).

For test peptide 1, CID at 20% NCE delivered a number of linker fragments, which were visible as singly and doubly negatively charged species (Fig. 4e). The HCD spectra, recorded at identical collision energies, looked similar to the CID spectra, yet with a larger number of linker fragments and neutral losses, whereas the loss of an intact peptide was not observed (Fig. S7e).


We evaluated two homobifunctional, amine-reactive, and MS-cleavable cross-linkers regarding their potential for a fast and facilitated analysis of cross-linked products. The BrPU linker, which is described for the first time, comprises two bromobenzoic acid groups that are connected by a central urea moiety. The unique structure of BrPU conserves the characteristic fragmentation pattern on collisional activation, exhibiting two doublets with a distinct mass difference of 25.979 u, that was previously found for our first urea-based cross-linking reagent [61, 63]. BrPU additionally imprints the bromine isotope pattern for all cross-linker-decorated fragments, greatly facilitating the identification of cross-linked species and their selection for MS/MS experiments. Additionally, we compared the fragmentation behavior of the BrPU linker with the recently introduced homobifunctional amine-reactive TEMPO-Bz linker that has been used for FRIPS studies. Comparative CID and HCD experiments were conducted in negative ESI mode with an Orbitrap Fusion mass spectrometer using five model peptides. Our studies reveal that linker fragmentation is the major pathway for both cross-linkers investigated, accompanied by peptide backbone fragmentation. In most cases, HCD yielded a higher number of sequence-specific fragment ions than CID. As hypothesized in a previous study, the presence of a cross-linked N-terminal aspartic acid residue seems to be the prerequisite for the unique loss of an intact peptide from the cross-linked products [56]. The novel BrPU linker combines a characteristic mass shift and isotope signature and therefore presents a more favorable combination for automated, fast, and reliable assignment of cross-linked products compared with the TEMPO-Bz linker.



A.S. and M.S. acknowledge financial support by the Deutsche Forschungsgemeinschaft (projects Si 867/15-2 and SCHA 871/7-2). A.S. also acknowledges funding by the state of Saxony-Anhalt. The tripeptides DAA, EAA, and VVA were a kind gift from Mike Schutkowski (Martin Luther University Halle-Wittenberg). The authors are indebted to Michael Götze for continuous improvement of the MeroX software and to Xiaohan Wang for excellent assistance with data analysis.

Supplementary material

13361_2017_1712_MOESM1_ESM.docx (8.6 mb)
ESM 1. ᅟ (DOCX 8793 kb)


  1. 1.
    Sinz, A.: Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. Mass Spectrom. Rev. 25, 663–682 (2006)Google Scholar
  2. 2.
    Petrotchenko, E.V., Borchers, C.H.: Crosslinking combined with mass spectrometry for structural proteomics. Mass Spectrom. Rev. 29, 862–876 (2010)Google Scholar
  3. 3.
    Sinz, A.: The advancement of chemical cross-linking and mass spectrometry for structural proteomics: from single proteins to protein interaction networks. Exp. Rev. Proteomics. 11, 733–743 (2014)Google Scholar
  4. 4.
    Sinz, A., Arlt, C., Chorev, D., Sharon, M.: Chemical cross-linking and native mass spectrometry: a fruitful combination for structural biology. Protein. Sci. 24, 1193–1209 (2015)Google Scholar
  5. 5.
    Leitner, A., Faini, M., Stengel, F., Aebersold, R.: Crosslinking and mass spectrometry: an integrated technology to understand the structure and function of molecular machines. Trends Biochem. Sci. 41, 20–32 (2016)Google Scholar
  6. 6.
    Tran, B.Q., Goodlett, D.R., Goo, Y.A.: Advances in protein complex analysis by chemical cross-linking coupled with mass spectrometry (CXMS) and bioinformatics. Biochim. Biophys. Acta. 1864, 123–129 (2016)Google Scholar
  7. 7.
    Yang, L., Zheng, C., Weisbrod, C.R., Tang, X., Munske, G.R., Hoopmann, M.R., Eng, J.K., Bruce, J.E.: In vivo application of photocleavable protein interaction reporter technology. J. Proteome. Res. 11, 1027–1041 (2012)Google Scholar
  8. 8.
    Kaake, R.M., Wang, X., Burke, A., Yu, C., Kandur, W., Yang, Y., Novtisky, E.J., Second, T., Duan, J., Kao, A., Guan, S., Vellucci, D., Rychnovsky, S.D., Huang, L.: A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells. Mol Cell Proteomics 13, 3533–3543 (2014)Google Scholar
  9. 9.
    Agou, F., Véron, M.: In vivo protein cross-linking. Methods Mol Biol. 1278, 391–405 (2015)Google Scholar
  10. 10.
    Green, N.S., Reisler, E., Houk, K.N.: Quantitative evaluation of the lengths of homobifunctional protein cross-linking reagents used as molecular rulers. Protein Sci. 10, 1293–1304 (2001)CrossRefGoogle Scholar
  11. 11.
    Kalkhof, S., Haehn, S., Paulsson, M., Smyth, N., Meiler, J., Sinz, A.: Computational modeling of laminin N-terminal domains using sparse distance constraints from disulfide bonds and chemical cross-linking. Proteins. 78, 3409–3427 (2010)CrossRefGoogle Scholar
  12. 12.
    Kahraman, A., Herzog, F., Leitner, A., Rosenberger, G., Aebersold, R., Malmstrom, L.: Cross-link guided molecular modeling with ROSETTA. PLoS One. 8, e73411 (2013)CrossRefGoogle Scholar
  13. 13.
    Hofmann, T., Fischer, A.W., Meiler, J., Kalkhof, S.: Protein structure prediction guided by crosslinking restraints - A systematic evaluation of the impact of the crosslinking spacer length. Methods. 89, 79–90 (2015)CrossRefGoogle Scholar
  14. 14.
    Schneider, M., Belsom, A., Rappsilber, J., Brock, O.: Blind testing of cross-linking/mass spectrometry hybrid methods in CASP11. Proteins: Structure, Function, and. Bioinformatics. 84, 152–163 (2016)Google Scholar
  15. 15.
    Leitner, A., Reischl, R., Walzthoeni, T., Herzog, F., Bohn, S., Forster, F., Aebersold, R.: Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography. Mol. Cell. Proteomics 11, M111.014126 (2012). doi: 10.1074/mcp.M111.014126
  16. 16.
    Fritzsche, R., Ihling, C.H., Götze, M., Sinz, A.: Optimizing the enrichment of cross-linked products for mass spectrometric protein analysis. Rapid Commun. Mass Spectrom. 26, 653–658 (2012)Google Scholar
  17. 17.
    Rappsilber, J., Mann, M., Ishihama, Y.: Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protocol. 2, 1896–1906 (2007)Google Scholar
  18. 18.
    Trnka, M.J., Baker, P.R., Robinson, P.J., Burlingame, A.L., Chalkley, R.J.: Matching cross-linked peptide spectra: only as good as the worse identification. Mol. Cell. Proteomics 13, 420–434 (2014)Google Scholar
  19. 19.
    Rasmussen, M.I., Refsgaard, J.C., Peng, L., Houen, G., Hojrup, P.: CrossWork: Software-assisted identification of cross-linked peptides. J. Proteomics. 74, 1871–1883 (2011)Google Scholar
  20. 20.
    Müller, D.R., Schindler, P., Towbin, H., Wirth, U., Voshol, H., Hoving, S., Steinmetz, M.O.: Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927–1934 (2001)Google Scholar
  21. 21.
    Petrotchenko, E.V., Olkhovik, V.K., Borchers, C.H.: Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes. Mol. Cell. Proteomics 4, 1167–1179 (2005)Google Scholar
  22. 22.
    Ihling, C., Schmidt, A., Kalkhof, S., Schulz, D.M., Stingl, C., Mechtler, K., Haack, M., Beck-Sickinger, A.G., Cooper, D.M., Sinz, A.: Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex. J. Am. Soc. Mass Spectrom. 17, 1100–1113 (2006)Google Scholar
  23. 23.
    Petrotchenko, E.V., Serpa, J.J., Borchers, C.H.: Use of a combination of isotopically coded cross-linkers and isotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks. Anal. Chem. 82, 817–823 (2010)Google Scholar
  24. 24.
    Brodie, N.I., Makepeace, K.A., Petrotchenko, E.V., Borchers, C.H.: Isotopically-coded short-range hetero-bifunctional photo-reactive crosslinkers for studying protein structure. J. Proteomics. 118, 12–20 (2015)Google Scholar
  25. 25.
    Spengler, B., Hester, A.: Mass-based classification (MBC) of peptides: highly accurate precursor ion mass values can be used to directly recognize peptide phosphorylation. J. Am. Soc. Mass Spectrom. 19, 1808–1812 (2008)Google Scholar
  26. 26.
    Ricks, A.M., Amster, I.J., Lie, C., Niehuser, S., Hernandez, H.: Mass defect labeling for enhanced protein identification. Abstr. Pap. Am. Chem. Soc. 229, U391–U391 (2005)Google Scholar
  27. 27.
    Valkenborg, D., Jansen, I., Burzykowski, T.: A model-based method for the prediction of the isotopic distribution of peptides. J. Am. Soc. Mass Spectrom. 19, 703–712 (2008)Google Scholar
  28. 28.
    Hall, M.P., Schneider, L.V.: Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation. Exp. Rev. Proteomics. 1, 421–431 (2004)Google Scholar
  29. 29.
    Kim, J.S., Song, S.U., Kim, H.J.: Simultaneous identification of tyrosine phosphorylation and sulfation sites utilizing tyrosine-specific bromination. J. Am. Soc. Mass Spectrom. 22, 1916–1925 (2011)Google Scholar
  30. 30.
    Li, C., Gawandi, V., Protos, A., Phillips, R.S., Amster, I.J.: A matrix-assisted laser desorption/ionization compatible reagent for tagging tryptophan residues. Eur. J. Mass. Spectrom. 12, 213–221 (2006)Google Scholar
  31. 31.
    Serpa, J.J., Parker, C.E., Petrotchenko, E.V., Han, J., Pan, J., Borchers, C.H.: Mass spectrometry-based structural proteomics. Eur. J. Mass Spectrom. 18, 251–267 (2012)Google Scholar
  32. 32.
    Sleno, L.: The use of mass defect in modern mass spectrometry. J. Mass Spectrom. 47, 226–236 (2012)Google Scholar
  33. 33.
    Steen, H., Mann, M.: Analysis of bromotryptophan and hydroxyproline modifications by high-resolution, high-accuracy precursor ion scanning utilizing fragment ions with mass-deficient mass tags. Analytical Chem. 74, 6230–6236 (2002)CrossRefGoogle Scholar
  34. 34.
    Ulbrich, A., Merrill, A.E., Hebert, A.S., Westphall, M.S., Keller, M.P., Attie, A.D., Coon, J.J.: Neutron-encoded protein quantification by peptide carbamylation. J. Am. Soc. Mass Spectrom. 25, 6–9 (2014)Google Scholar
  35. 35.
    Hoffman, L., Griffin, P., Fechheimer, M., Petrotchenko, E., Borchers, C., Amster, J.: Development of a mass-spectrometry identifiable crosslinker and application to a 34kDa-actin protein system. Proceedings of the 57th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia, PA (2009)Google Scholar
  36. 36.
    Miyagi, M., Nakao, M., Nakazawa, T., Kato, I., Tsunasawa, S.: A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides. Rapid Commun. Mass Spectrom. 12, 603–608 (1998)Google Scholar
  37. 37.
    Kim, J.S., Shin, M., Song, J.S., An, S., Kim, H.J.: C-terminal de novo sequencing of peptides using oxazolone-based derivatization with bromine signature. Anal. Biochem. 419, 211–216 (2011)Google Scholar
  38. 38.
    Nam, J., Kwon, H., Jang, I., Jeon, A., Moon, J., Lee, S.Y., Kang, D., Han, S.Y., Moon, B., Oh, H.B.: Bromine isotopic signature facilitates de novo sequencing of peptides in free-radical-initiated peptide sequencing (FRIPS) mass spectrometry. J. Mass Spectrom. 50, 378–387 (2015)CrossRefGoogle Scholar
  39. 39.
    Kim, J.S., Kim, J., Oh, J.M., Kim, H.J.: Tandem mass spectrometric method for definitive localization of phosphorylation sites using bromine signature. Anal. Biochem. 414, 294–296 (2011)Google Scholar
  40. 40.
    Shi, Y., Bajrami, B., Morton, M., Yao, X.: Cyclophosphoramidate ion as mass defect marker for efficient detection of protein serine phosphorylation. Anal. Chem. 80, 7614–7623 (2008)Google Scholar
  41. 41.
    Mao, Y., Zamdborg, L., Kelleher, N.L., Hendrickson, C.L., Marshall, A.G.: Identification of phosphorylated human peptides by accurate mass measurement alone. Int. J. Mass Spectrom. 308, 357–361 (2011)Google Scholar
  42. 42.
    McLuckey, S.A., Goeringer, D.E.: Slow heating methods in tandem mass spectrometry. J. Mass Spectrom. 32, 461–474 (1997)Google Scholar
  43. 43.
    Qi, Y., Volmer, D.A.: Structural analysis of small to medium-sized molecules by mass spectrometry after electron-ion fragmentation (ExD) reactions. Analyst. 141, 794–806 (2016)CrossRefGoogle Scholar
  44. 44.
    Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Nat. Acad. Sci. USA. 101, 9528–9533 (2004)Google Scholar
  45. 45.
    Frese, C.K., Altelaar, A.F.M., van den Toorn, H., Nolting, D., Griep-Raming, J., Heck, A.J.R., Mohammed, S.: Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higher-energy collision dissociation tandem mass spectrometry. Anal. Chem. 84, 9668–9673 (2012)Google Scholar
  46. 46.
    Giese, S.H., Belsom, A., Rappsilber, J.: Optimized fragmentation regime for diazirine photo-cross-linked peptides. Anal. Chem. 88, 8239–8247 (2016)Google Scholar
  47. 47.
    Arlt, C., Götze, M., Ihling, C.H., Hage, C., Schäfer, M., Sinz, A.: Integrated workflow for structural proteomics studies based on cross-linking/mass spectrometry with an MS/MS cleavable cross-linker. Anal. Chem. 88, 7930–7937 (2016)Google Scholar
  48. 48.
    Syka, J.E.P., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Nat. Acad. Sci. USA. 101, 9528–9533 (2004)Google Scholar
  49. 49.
    Zubarev, R.A., Zubarev, A.R., Savitski, M.M.: Electron capture/transfer versus collisionally activated/induced dissociations: solo or duet? J. Am. Soc. Mass Spectrom. 19, 753–761 (2008)Google Scholar
  50. 50.
    Chowdhury, S.M., Du, X.X., Tolic, N., Wu, S., Moore, R.J., Mayer, M.U., Smith, R.D., Adkins, J.N.: Identification of cross-linked peptides after click-based enrichment using sequential collision-induced dissociation and electron transfer dissociation tandem mass spectrometry. Anal. Chem. 81, 5524–5532 (2009)Google Scholar
  51. 51.
    Liu, F., Rijkers, D.T., Post, H., Heck, A.J.: Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat. Methods. 12, 1179–1184 (2015)Google Scholar
  52. 52.
    Hodyss, R., Cox, H.A., Beauchamp, J.L.: Bioconjugates for tunable peptide fragmentation: free radical initiated peptide sequencing (FRIPS). J. Am. Chem. Soc. 127, 12436–12437 (2005)Google Scholar
  53. 53.
    Gao, J., Thomas, D.A., Sohn, C.H., Beauchamp, J.L.: Biomimetic reagents for the selective free radical and acid-base chemistry of glycans: application to glycan structure determination by mass spectrometry. J. Am. Chem. Soc. 135, 10684–10692 (2013)Google Scholar
  54. 54.
    Oh, H.B., Moon, B.: Radical-driven peptide backbone dissociation tandem mass spectrometry. Mass Spectrom. Rev. 34, 116–132 (2015)CrossRefGoogle Scholar
  55. 55.
    Lee, M., Kang, M., Moon, B., Oh, H.B.: Gas-phase peptide sequencing by TEMPO-mediated radical generation. Analyst. 134, 1706–1712 (2009)Google Scholar
  56. 56.
    Hage, C., Ihling, C.H., Götze, M., Schäfer, M., Sinz, A.: Dissociation behavior of a TEMPO-active ester cross-linker for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) in negative ESI-MS. J. Am. Soc. Mass Spectrom. 28, 56–68 (2017)Google Scholar
  57. 57.
    Sinz, A.: Divide and conquer: cleavable cross-linkers to study protein conformation and protein-protein interactions. Anal. Bioanal. Chem. 409, 33–44 (2017)Google Scholar
  58. 58.
    Soderblom, E.J., Goshe, M.B.: Collision-induced dissociative chemical cross-linking reagents and methodology: applications to protein structural characterization using tandem mass spectrometry analysis. Anal. Chem. 78, 8059–8068 (2006)Google Scholar
  59. 59.
    Soderblom, E.J., Bobay, B.G., Cavanagh, J., Goshe, M.B.: Tandem mass spectrometry acquisition approaches to enhance identification of protein-protein interactions using low-energy collision-induced dissociative chemical crosslinking reagents. Rapid Commun. Mass Spectrom. 21, 3395–3408 (2007)Google Scholar
  60. 60.
    Argo, A.S., Shi, C., Liu, F., Goshe, M.B.: Performing protein crosslinking using gas-phase cleavable chemical crosslinkers and liquid chromatography-tandem mass spectrometry. Methods. 89, 64–73 (2015)CrossRefGoogle Scholar
  61. 61.
    Müller, M.Q., Dreiocker, F., Ihling, C.H., Schäfer, M., Sinz, A.: Cleavable cross-linker for protein structure analysis: reliable identification of cross-linking products by tandem MS. Anal. Chem. 82, 6958–6968 (2010)Google Scholar
  62. 62.
    Müller, M.Q., Dreiocker, F., Ihling, C.H., Schäfer, M., Sinz, A.: Fragmentation behavior of a thiourea-based reagent for protein structure analysis by collision-induced dissociative chemical cross-linking. J. Mass Spectrom. 45, 880–891 (2010)Google Scholar
  63. 63.
    Müller, M.Q., Zeiser, J.J., Dreiocker, F., Pich, A., Schäfer, M., Sinz, A.: A universal matrix-assisted laser desorption/ionization cleavable cross-linker for protein structure analysis. Rapid Commun. Mass Spectrom. 25, 155–161 (2011)Google Scholar
  64. 64.
    Kao, A.H., Chiu, C.L., Vellucci, D., Yang, Y.Y., Patel, V.R., Guan, S.H., Randall, A., Baldi, P., Rychnovsky, S.D., Huang, L.: Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes. Mol. Cell Proteomics. 10, 1–17 (2011)Google Scholar
  65. 65.
    Kandur, W.V., Kao, A., Vellucci, D., Huang, L., Rychnovsky, S.D.: Design of CID-cleavable protein cross-linkers: identical mass modifications for simpler sequence analysis. Org. Biomol. Chem. 13, 9793–9807 (2015)Google Scholar
  66. 66.
    Burke, A.M., Kandur, W., Novitsky, E.J., Kaake, R.M., Yu, C., Kao, A., Vellucci, D., Huang, L., Rychnovsky, S.D.: Synthesis of two new enrichable and MS-cleavable cross-linkers to define protein-protein interactions by mass spectrometry. Org. Biomol. Chem. 13, 5030–5037 (2015)Google Scholar
  67. 67.
    Gardner, M.W., Brodbelt, J.S.: Preferential cleavage of N-N hydrazone bonds for sequencing bis-arylhydrazone conjugated peptides by electron transfer dissociation. Anal. Chem. 82, 5751–5759 (2010)Google Scholar
  68. 68.
    Trnka, M.J., Burlingame, A.L.: Topographic studies of the GroEL-GroES chaperonin complex by chemical cross-linking using diformyl ethynylbenzene: the power of high resolution electron transfer dissociation for determination of both peptide sequences and their attachment sites. Mol. Cell. Proteomics. 9, 2306–2317 (2010)Google Scholar
  69. 69.
    Gardner, M.W., Vasicek, L.A., Shabbir, S., Anslyn, E.V., Brodbelt, J.S.: Chromogenic cross-linker for the characterization of protein structure by infrared multiphoton dissociation mass spectrometry. Anal. Chem. 80, 4807–4819 (2008)Google Scholar
  70. 70.
    Petrotchenko, E.V., Xiao, K., Cable, J., Chen, Y., Dokholyan, N.V., Borchers, C.H.: BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics. Mol. Cell. Proteomics. 8, 273–286 (2009)Google Scholar
  71. 71.
    Ihling, C., Falvo, F., Kratochvil, I., Sinz, A., Schäfer, M.: Dissociation behavior of a bifunctional tempo-active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry. J. Mass Spectrom. 50, 396–406 (2015)Google Scholar
  72. 72.
    Falvo, F., Fiebig, L., Dreiocker, F., Wang, R., Armentrout, P.B., Schafer, M.: Fragmentation reactions of thiourea- and urea-compounds examined by tandem MS-, energy-resolved CID experiments, and theory. Int. J. Mass Spectrom. 330, 124–133 (2012)Google Scholar

Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  • Christoph Hage
    • 1
  • Francesco Falvo
    • 2
    • 3
  • Mathias Schäfer
    • 2
  • Andrea Sinz
    • 1
  1. 1.Institute of PharmacyMartin Luther University Halle-WittenbergHalle (Saale)Germany
  2. 2.Department of ChemistryUniversity of CologneCologneGermany
  3. 3.Eurofins Umwelt West GmbHWesselingGermany

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