Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors.


Introduction
(Photo-) chemical cross-linking combined with mass spectrometry (MS) has evolved as an alternative method to obtain low-resolution three-dimensional (3D) structural information of proteins and protein complexes. [1][2][3][4][5][6][7][8][9] The cross-linking/MS approach allows deriving structural information by covalently connecting pairs of functional groups in the protein(s) under investigation. This work follows up on a previous study, in which the distances bridged by different cross-linkers as well as an incorporated photo-reactive amino acid had served as "molecular rulers" to map the 3D-structures of free and ligand-bound peroxisome proliferatoractivated receptor alpha (PPAR-α). Using chemical cross-linking/MS, we had been able to monitor conformational changes in the ligand-binding domain (LBD) of PPAR-α upon ligand binding. [8] In the present work, we aim to monitor conformational changes in the PPAR-β/δ isoform. For this, we applied the amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate (BS 2 G; Fig  1A) that bridges Cα-Cα distances up to 27 Å. [10,11] BS 2 G is an N-hydroxysuccinimide (NHS) ester that connects lysines-as well as the N-terminus-in a protein, but it also possesses a tendency to react with serines, threonines, and tyrosines. [12] As an additional amine-reactive cross-linker we used the in-house synthesized urea-cross-linker ( Fig 1B). [13] This linker possesses unique properties for an automated identification of cross-linked products based on its characteristic fragmentation patterns that are created upon collision-induced dissociation (CID)-tandem MS conditions. Also, cross-linking experiments were performed with the zerolength cross-linker DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; Fig 1C) that is able to connect amines (N-terminus and lysines) with carboxyl groups MS/MS-cleavable amine-reactive urea-linker; cleavable bonds are indicated in red; the fragments created under MS/MS conditions are denoted as "Bu" and "BuUr" according to [15]. (C) Reaction mechanism of the zero-length cross-linker DMTMM, connecting carboxyl and amine groups in proteins. (glutamic acid and aspartic acid). In a recent study, DMTMM was employed as activating reagent to couple carboxyl groups in proteins by a hydrazide cross-linker. [14] In addition, we performed photo-cross-linking experiments by incorporating the unnatural photo-activatable amino acid para-benzoyl-L-phenylalanine (Bpa) at specific positions into the LBD of PPAR-β/δ. [16] The benzophenone group of Bpa is activated by UV-A light and can potentially insert into CH, NH, SH and OH groups of all 20 proteinogenic amino acids (Fig 2); yet, it has been shown that Bpa possesses a certain preference towards methionines. [17,18] PPARs are ligand-activated transcription factors that belong to the nuclear receptor protein family. So far, three subtypes of PPARs (α, β/δ, γ) have been identified. [19][20][21] They are composed of a DNA-binding domain (DBD) and a ligand-binding domain (LBD; for amino acid sequence, please see S1 Fig). PPARs are activated by fatty acids and eicosanoids, but also by a number of low-molecular weight compounds. [22][23][24][25][26] After activation, PPARs form heterodimers with the retinoid X receptor and bind to specific DNA sequences. [27,28] In general, PPAR-α promotes fatty acid catabolism in the liver and the skeletal muscle, while PPAR-γ regulates fatty acid storage in adipose tissues. [29][30][31] PPAR-β/δ is expressed ubiquitously [32] and is involved in fatty acid catabolism, [33] cell differentiation, [34] and cancer. [35] Due to its complex roles in the human metabolism, PPAR-β/δ is an attractive target for drug design.
Here, we study the conformational changes in the LBD as well as full-length PPAR-β/δ upon binding of the agonists GW0742 (K D = 0.4 nM, EC 50 = 1 nM) and GW1516 (K D = 1.1 nM, EC 50 = 1 nM) (Fig 3). [22] After (photo-) chemical cross-linking, enzymatic digestion, and MS analysis of the cross-linked peptides, the distance constraints obtained allowed us to study the conformational changes upon ligand binding in PPAR-β/δ. The identified cross-links in the DBD and the LBD were in good agreement with published NMR and X-ray 3D-structures (pdb entries 2ENV and 3TKM; Fig 4). [36] With the (photo-) cross-linking/MS approach, we obtained detailed structural information mainly from the flexible N-terminal region and the hinge region of full-length PPAR-β/δ upon ligand binding.

Cross-Linking Experiments
TEV protease was added (5-10 U) to PPAR-β/δ LBD wildtype and variants as well as fulllength PPAR-β/δ to remove the tag (Strep-tag II for PPAR-β/δ LBD; (His) 6 -tag for full-length PPAR-β/δ). After cleavage (6°C, overnight) all samples were washed three times with buffer A (see Protein Expression and Purification) using an Amicon centrifugation unit (10 kDa). Samples were adjusted to a protein concentration of 5 μM and immediately used for (photo-) cross-linking experiments. For photo-cross-linking, the PPAR-β/δ LBD variants F180Bpa and Y443Bpa were mixed with the agonist GW0742 or GW1516 in DMSO to give a 20-fold molar excess of ligand over protein. As a control, one sample was mixed with DMSO without the addition of ligand. After 30 min at 4°C, the solutions were irradiated with UV-A light (365 nm; 8 J/cm 2 ) to activate the photo-reactive amino acid Bpa (Scheme 1). Afterwards, samples were analyzed by SDS−PAGE or subjected to in-solution digestion with trypsin or a trypsin/GluC mixture. Bands of interest were excised from SDS-gels and in-gel digested with trypsin according to an existing protocol. [37] After enzymatic digestion, the peptide mixtures were immediately analyzed by LC/MS/MS. PPAR-β/δ LBD and full-length PPAR-β/δ were mixed with both agonists as described above. After incubation on ice, the PPAR-β/δ LBD was mixed either with DMTMM (8000-fold molar excess over protein), BS 2 G-D 0 /D 4 (100-or 200-fold molar excess over protein) or the urea-linker (100-or 200-fold molar excess over protein) for 2 hrs on ice. Samples were either quenched with ammonium bicarbonate (BS 2 G-D 0 /D 4 and urea-linker) to a final concentration of 20 mM or immediately mixed with Laemmli buffer and analyzed by SDS-PAGE (DMTMM). Full-length PPAR-β/δ was mixed with BS 2 G-D 0 /D 4 to give a 200-fold molar excess of cross-linker over the protein. After 2 hrs on ice, samples were quenched as described above.

Visualization of Cross-links
All 3D-structures were created with PyMOL (0.99rc6). Circos plots were created with Circos (0.67-7). [40] Results The X-ray structure of PPAR-β/δ LBD has been determined in complex with the agonist GW0742 (pdb entry 3TKM). [36] Yet, no high-resolution structural information is available to date on how GW1516 interacts with PPAR-β/δ. Given that both agonists differ only by the presence of a fluorine atom, similar binding modes were expected for both ligands. To clarify this, we employed the chemical cross-linking/MS approach using a variety of external crosslinkers as well as the incorporation of the photo-reactive amino acid Bpa. For the latter, two Bpa variants of PPAR-β/δ LBD (amino acids 167-443) were expressed in E.coli cells using the method developed by Schultz and coworkers. [16] PPAR-β/δ LBD variants were individually transformed into E. coli cells carrying a Bpa-specific suppressor tRNA and an aminoacyl-tRNA synthetase that allows the incorporation of Bpa in place of the natural amino acid via the introduced TAG stop codon. In PPAR-β/δ LBD, either Phe-180 or Tyr-443 was replaced by the photo-reactive amino acid Bpa: Phe-180 is located on Arm II in helix 1, while Tyr-443 is located on Arm I of activation function helix 2 (AF2, helix 12) ( Fig 4A). As both helices are prone to conformational changes upon ligand binding they are ideal targets for the incorporation of the photo-reactive amino acid Bpa.

Purification of PPAR-β/δ LBD and Variants F180Bpa and Y443Bpa
PPAR-β/δ LBD and Bpa variants were purified as N-terminally Strep II-tagged proteins via affinity chromatography. Pure protein was obtained after TEV protease cleavage (~13 μg/g cells for PPAR-β/δ LBD and~6.5 μg/g cells for PPAR-β/δ LBD variants). The identity of the PPAR-β/δ LBD variants and the incorporation of Bpa at the desired positions were confirmed by in-gel digestion and LC/MS/MS analysis.

Cross-linking with Amine-Reactive and Zero-Length Linkers
After purification of PPAR-β/δ LBD, we performed cross-linking experiments with the homobifunctional cross-linker BS 2 G that bridges Cα-Cα distances up to 27 Å (Fig 1A). Cross-links identified between lysines-and to a certain extent also with serines, threonines, and tyrosinesallow deducing conformational information in PPAR-β/δ. We identified a number of crosslinks in the absence, but also in the presence of ligands (S1 Table). All cross-links identified in free PPAR-β/δ LBD were in accordance with the published X-ray structure (pdb entry 3TKM). Upon agonist binding, additional cross-links were identified between the N-terminus of the LBD (corresponding to Gly-167) and the flexible O-loop (for agonist GW1516) as well as the activation function helix 2 (for agonist GW0742) (Fig 4A). Interestingly, these cross-links were not identified in free PPAR-β/δ LBD and indicate conformational changes upon binding of either of the agonists. All cross-links found for BS 2 G are presented in Fig 5. Additional cross-linking experiments were performed using the MS/MS cleavable aminereactive urea-linker (Fig 1B) that allows an automated analysis of cross-links via its characteristic fragmentation pattern. [13,15,39] As such, a cross-link between the N-terminus of the LBD (Gly-167) and Lys-198 in PPAR-β/δ was unambiguously identified based on the specific fragmentation pattern of the urea-linker and intense backbone cleavage of the connected peptides ( Fig 6A). The urea-linker is an amine-reactive homobifunctional cross-linker that bridges Cα-Cα distances up to 30 Å, which is only slightly longer than the distances BS 2 G can connect. As such, it is not surprising that for PPAR-β/δ LBD most of the cross-linking sites found in experiments with BS 2 G were also identified with the urea-linker (S2 Table).
To allow carboxylic acid side chains in PPAR-β/δ LBD to participate in the cross-linking reaction, we also employed the zero-length cross-linker DMTMM (Fig 1C). DMTMM crosslinks spatially neighbored amine groups (N-terminus and lysine residues) and carboxyl groups (aspartic and glutamic acid residues) and thus yields complementary short-range information compared to the homobifunctional amine-reactive linkers. With DMTMM, similar cross-links were identified in PPAR-β/δ LBD in the presence as well as in the absence of ligands (S2 Table). MS/MS data of one exemplary cross-link are shown in Fig 6B. In GW0742-bound PPAR-β/δ LBD, two additional cross-links were identified, connecting the N-and the C-termini as well as the N-terminus (Gly167) and Glu-331 of the LBD. Both cross-links cannot be explained by the published X-ray structure. In order to be cross-linked by DMTMM, the respective amino acid side chains have to come into close spatial neighborhood indicating that PPAR-β/δ LBD adopts a specific conformation in solution after ligand binding.

Photo-Cross-linking with Bpa Variants
To obtain complementary structural information, the PPAR-β/δ LBD variants F180Bpa and Y443Bpa were produced by genetic engineering. [16] In each of the two variants, the photoreactive amino acid Bpa was specifically introduced at a defined position in different flexible regions of PPAR-β/δ LBD to optimize monitoring conformational changes upon ligand binding ( Fig 4A). Photo-cross-linking experiments were conducted in the presence and the absence of agonists GW0742 and GW1516 by irradiation with UV-A light. After the photo-cross-linking reaction, PPAR-β/δ LBD variants were enzymatically digested and the resulting peptide mixtures were analyzed by LC/ESI-MS/MS yielding several cross-links (S3 Table). For PPARβ/δ variant F180Bpa, one cross-link was found in the absence as well as in the presence of agonists (Fig 7). For variant PPAR-β/δ Y443Bpa, two cross-links were identified in the absence, while one was identified in the presence of ligands (S3 Table). All cross-links identified with our complementary approaches are visualized as Circos plots (Fig 8A-8C, S2-S13 Figs). [40] Photo-cross-links identified in PPAR-β/δ variants F180Bpa and Y443Bpa were comparable for free and agonist-bound protein. In ligand-free PPAR-β/δ variant Y443Bpa, an additional cross-link between Bpa-443 and Ile-330 (located in the N-terminal region of helix 8) was identified, which perfectly matches the published X-ray structure (Fig 4A).

Cross-linking with Full-Length PPAR-β/δ
We performed additional cross-linking experiments with full-length PPAR-β/δ comprising the DBD and the LBD (S1 Fig) to rule out that some cross-links found for PPAR-β/δ LBD might be induced by the artificially created N-terminus, but in fact do not represent the native structure of PPAR-β/δ. For full-length PPAR-β/δ, a high number of cross-links were identified in the flexible hinge region connecting DBD and LBD as well as in the flexible N-terminal region-both in  Table). All unambiguously identified cross-links were in good agreement with published high-resolution 3D-structures of the DBD and the LBD. Several of these cross-links were found between the flexible N-terminal and hinge regions and the DBD or within the DBD (Fig 8). As such, the N-terminus of full-length PPAR-β/δ was  cross-linked-both in the presence and in the absence of ligands-to Lys-198 in the LBD as well as to a number of amino acids in the DBD (amino acids 73-147), i.e., Lys-122, Lys-107, and Lys-126. Interestingly, only in the presence of GW1516 an additional cross-link between the Nterminus and Lys-423 was found (Fig 8C).

Structures of PPAR-β/δ LBD
Chemical cross-linking with the amine-reactive cross-linkers BS 2 G and the urea-linker yielded a high number of cross-links in free as well as in ligand-bound PPAR-β/δ LBD (Table 1, S1 and S2 Tables). After ligand binding, several cross-links were identified between the N-terminus (corresponding to Gly-167) of the isolated LBD and various lysines in PPAR-β/δ LBD. Some of these cross-links cannot be explained by the X-ray structure of GW0742-bound PPAR-β/δ LBD (pdb 3TKM; Fig 5A-5C), taking the distances into account the cross-linkers can bridge (up to 30 Å). They can, however, be explained by a large degree of flexibility in the N-terminal region of the LBD where Gly-167 is located. As such, in the ligand-bound-state of PPAR-β/δ LBD, cross-links were identified between the N-terminus (Gly-167) and Lys-423/424 (C-terminal region of helix 11) and Lys-324 (N-terminal region of helix 8) (Fig 5B and 5C). With the agonist GW0742, cross-links between the N-terminus of the LBD (Gly-167) and Lys-440 (AF2) as well as between Lys-197/198 (helix 2) and Lys-324 (N-terminal region of helix 8) were identified (Fig 5B). In the GW1516-bound state, a cross-link between the N-terminal Gly-167 and Lys-231, located on the flexible O-loop, was identified (S1 Table). As no diffraction data are available for parts of the omega loop in the high-resolution X-ray structure, the location of Lys-   Table 1. Comparison of identified (photo)-cross-links in full-length PPAR-β/δ and PPAR-β/δ LBD. For the LBD, cross-links are summarized for all reagents used in this study, while for full-length PPAR-β/δ only BS 2 G cross-links are presented; { denotes the N-terminus of the protein; } denotes the C-terminus of the protein.

PPAR-β/δ ligand-binding domain (LBD) PPAR-β/δ full-length
Cross-linked amino acids Free GW0742 GW1516 Cross-linked amino acids Free GW0742 GW1516 231 is ambiguous and as such, the respective residue cannot be assigned in Fig 5C. This crosslink might be explained by a large conformational change in the O-loop after ligand binding, bringing the N-terminus of the LBD into close distance to the O-loop. Also, in both ligand-bound states, cross-links were found between the N-terminal Gly-167 of the LBD and Lys-404 (N-terminal region of helix 11) and Lys-267 (C-terminal region of helix 4) (Fig 5B and 5C) that are in agreement with the distances the cross-linkers can bridge. The fact that both cross-links were not identified in free PPAR-β/δ LBD suggests the presence of a different stabilized conformation upon ligand binding.
Additional cross-linking experiments performed with the zero-length cross-linker DMTMM that delivers valuable short-range information resulted in a high number of crosslinks, both in the free and in the ligand-bound state of PPAR-β/δ LBD (S2 Table). In all states, a cross-link was identified between Gly-167 and Glu-264, located in the C-terminal region of helix 4. Interestingly, this helix was also cross-linked by BS 2 G in the ligand-bound state (Lys-267) indicating that Gly-167 comes into close distance to helix 4 and is thus cross-linked by BS 2 G and the zero-length cross-linker DMTMM. Two additional cross-links between Gly-167 and the C-terminus as well as between Gly-167 and Glu-331 (located on helix 8) were identified in the GW0742-bound state. Both cross-links cannot be explained by the published X-ray structural data, again confirming the stabilization of a specific conformation where the N-terminus of the LBD comes into close spatial neighborhood to helices 4 and 8 ( Fig 4A). Yet, with GW1516 these cross-linking sites were not identified, possibly indicating different binding modes for the two agonists GW0742 and GW1516.
The incorporation of the photo-reactive amino acid Bpa at two defined positions into PPAR-β/δ LBD, replacing either Phe-180 or Tyr-443, followed by UV-irradiation yielded only a few cross-links, underlining the selectivity of this strategy. Most of the cross-links were identified in free as well as in ligand-bound states of PPAR-β/δ LBD. For PPAR-β/δ variant F180Bpa, one cross-linking site was identified, connecting Ile-379 on helix 10 with Bpa-180 (Fig 4A). The cross-link between the Bpa-443, located on AF2, and Ile-330, located on helix 8 (Fig 4A), was found exclusively in the ligand-free state of PPAR-β/δ variant Y443Bpa. No cross-links were identified between AF2 and the flexible O-loop, indicating that the O-loop and the AF2 are not oriented towards each other, which matches the published X-ray structure (Fig 4A).

Structures of Full-Length PPAR-β/δ
To complement our results obtained with the LBD of PPAR-β/δ, we performed cross-linking experiments with full-length PPAR-β/δ using the amine-reactive cross-linker BS 2 G. We identified a large number of cross-links in the hinge region between DBD and LBD, in the flexible Nterminal region, and within the DBD (Fig 8). A cross-link identified in the ligand-free state between Lys-155 (located in the hinge region) and Lys-404 (located on helix 11) of full-length PPAR-β/δ points to the same conformation as the cross-link found between Gly-167 and Lys- 404 in PPAR-β/δ LBD. The fact that this cross-link was identified both in the ligand-bound state of PPAR-β/δ LBD as well as in the ligand free-state of full-length PPAR-β/δ suggests an overall presence of this specific conformation. It might be speculated that in the truncated form of PPAR-β/δ, comprising only the LBD, the N-terminal region and helix 1 are more flexible than in full-length PPAR-β/δ, resulting in more pronounced conformational changes upon ligand binding than in full-length PPAR β/δ. In the GW1516-bound state, a cross-link was identified between the N-terminus and Lys-423 (located on helix 11 in the LBD) in full-length PPAR-β/δ. This suggests that after binding of GW1516, the Nand C-termini of PPAR-β/δ come into close neigborhood (Fig 8C). The fact that this cross-link was specifically identified in GW1516-bound PPAR-β/δ, however, does not necessarily indicate that is it not present in GW0742-bound protein. Clearly, more cross-linking experiments are required to support the finding that a specific conformation is induced in full-length PPAR-β/δ only in the presence of GW1516.
Our cross-linking results yield first structural insights into the conformational changes of full-length PPAR-β/δ upon ligand binding. In the past years, crystal structures of three nuclear receptors have emerged showing interfacial coupling between the DBDs and LBDs. [41][42][43][44] Our data confirm and extend these high-resolution structural data, indicating interactions between DBD and LBD in PPAR-β/δ in solution (Fig 8). We mapped the cross-links found for full-length PPAR-β/δ into the published X-ray structure of ligand-bound PPAR-γ, co-crystallized with DNA response element, coactivator peptides, and RXR-α (pdb 3DZU) [41] (S14 Fig). All cross-links between DBD and LBD as well as those connecting the flexible hinge region with DBD and LBD in PPAR-β/δ proved to be in good agreement with the X-ray structure. One slightly longer cross-link (40.8 Å) between the hinge region and helix 11 (LBD) can easily be explained by the inherent flexibility of the hinge region. Moreover, our cross-linking data were able to deliver structural information of the highly flexible N-terminus of PPAR-β/δ, for which no structural data are available in the PPAR-γ structure.
Conclusively, the cross-linking/MS approach proved highly advantageous to study nuclear receptors, enabling us to reveal the interplay between DBD and LDB in PPAR-β/δ. We envision that chemical cross-linking/MS, combined with other structural, biophysical, and cell-based studies will enhance our current knowledge of how PPARs function and how conformational changes occur when activating ligands are present.

Conclusions & Outlook
The cross-linking/MS approach revealed different conformations of PPAR-β/δ LBD and fulllength PPAR-β/δ in solution, stabilizing one specific conformation through ligand binding. To our best knowledge, we performed the first studies addressing the structure of full-length PPAR-β/δ upon ligand binding, revealing the interplay between DBD and LDB in free and ligand-bound PPAR-β/δ. Our cross-linking data were able to deliver structural information also from the highly flexible N-terminus of PPAR-β/δ, for which no structural data are available in the PPAR-γ structure. Moreover, a close distance between the Nand C-terminal regions was observed for full-length PPAR-β/δ in the presence of GW1516. Further cross-linking experiments are planned for PPAR-β/δ in the presence of DNA response element and RXR to complement existing high-resolution 3D-structural data. 13-bound PPAR-γ, co-crystallized with the DNA response element (PPRE), coactivator peptides (NCOA2), and RXR-α (pdb 3DZU). Crosslinks identified in full-length PPAR-β/δ are mapped in the crystal structure of PPAR-γ (shown in green). RXR-α and NCOA2 are shown in grey, PPRE in light blue, DBD of PPAR-γ in red, the hinge region in wheat, the LBD of PPAR-γ in orange, coactivator in pale cyan, and the agonist BVT.13 in magenta. (DOCX) S1 Table. Summary of identified BS 2 G cross-links in experiments with BS 2 G in PPAR β/δ LBD. Cross-linked peptides are summarized; masses of cross-linked products with the crosslinker BS 2 G-D 0 (light) /D 4 (heavy) are given; { denotes N-terminus of the protein; n denotes deamidated asparagine (corresponding to D); m denotes methionine oxidation. (DOCX) S2 Table. Summary of identified cross-links in experiments with BS 2 G in PPAR β/δ LBD, using DMTMM or the urea-linker. Cross-linked peptides are summarized; masses of crosslinked products with the cross-linkers (DMTMM or urea-linker) are given; { denotes N-terminus of the protein; n denotes deamidated asparagine (corresponding to D); q denotes glutamine deamidation (corresponding to E); m denotes methionine oxidation. (DOCX) S3 Table. Summary of identified photo-cross-links in PPAR β/δ variants F180Bpa and Y443Bpa. Photo-cross-linked peptides are summarized; masses of cross-linked products with the photo-reactive amino acid Bpa are given; { denotes N-terminus of the protein;} denotes Cterminus of the protein; q denotes glutamine deamidation (corresponding to E); m denotes methionine oxidation. (DOCX) S4 Table. Summary of identified BS 2 G cross-links in full-length PPAR β/δ. Cross-linked peptides are summarized; masses of cross-linked products with the cross-linker BS 2 G-D 0 (light) /D 4 (heavy) are given; { denotes N-terminus of the protein;} denotes C-terminus of the protein; q denotes glutamine deamidation (corresponding to E); n: denotes asparagine deamidation (corresponding to D); B denotes carbamidomethylation of cysteine; m denotes methionine oxidation; X denotes Bpa. (DOCX)