Crosslinking mass-spectrometry data analysis

We performed XL-MS/MS experiment in order to investigate the interaction map of the MMH complex and enhance our understanding of the domains that are in close proximity. For XL-MS/MS analysis, the BS3 _{crosslinker was used to crosslink both the}

MMH and MMHG complexes (Fig 4-13). Briefly, the MMH complex were overexpressed and purified from EXPI293 cells, then ~ 40 µg of FLAG purified complex was mixed with BS3 _{for 30 min and then the crosslinkers were quenched (see materials}

and methods). Of note, MMH crosslinking with DSS failed (data not shown here) so we switched to BS3 _{and used only a 30-min incubation, as we did before for the MR}

complex (161). Because the 30-min incubation yielded a significant number of crosslinked peptides, we did not do any further optimization. Next, cross-linked complexes were digested with trypsin and Lys-C proteases, and the resulting mixtures run on size exclusion chromatography (SEC), in order to enrich for larger peptides, which are more likely to be the crosslinked ones that are being sought. The crosslinked peptides were then analysed using a Q-Exactive LC-MS/MS instrument.

Fig 4-13. BS3 _{cross-linked complexes. (A) Sypro-Ruby stained SDS-PAGE}

showing a band over 200 kDa after crosslinking the MMH complex. (B) Sypro-Ruby stained SDS-PAGE showing a band over 200 kDa after crosslinking the MMHG complex. Five percent of the crosslinked materials was run onto the SDS-PAGE. Lane M is the Mark12 ladder. Red lines schematically indicate the BS3 _{crosslinker that crosslinks the}

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The pLink software package [223] was used for crosslinking mass spectrometry data analysis. Identification of spectra and peptide pairs were performed under conditions of 5% FDR control. XL-MS/MS data provide information about cross-linked, mono- linked, loop-linked, and regular peptides. Monolink or deadend peptides are generated from XLs that have only one functional group because the other group is hydrolyzed during sample processing. However, looplink peptides form a reaction in which both functional groups of the cross-linker react with lysine residues that exist in the same peptide after enzymatic digestion. Inter XLs connect peptides from different proteins, providing information about subunit proximity in a complex whereas intra XLs connect lysine residues on two different peptides from the same protein.

XL-MS/MS analysis showed the same data for both MMH and MMHG complexes and henceforth we only discuss the MMH XL-MS/MS data. Table 2 shows the XL-MS/MS data for MMH complex, in the format of spectra and peptides. It indicates that only 25% of the identified peptides were BS3 _{cross-linked peptides (inter- and intra- XLs),}

while the majority (46%) were regular peptides. About ~20% and ~7% of the peptides were mono-linked and loop-linked peptides. The loop-linked and mono-linked cannot provide any distance information between proteins of the complex, but can yield important information regarding which lysines are exposed and available for crosslinking. One reason for dominance of normal peptides is that, even with enrichment strategies such as size exclusion chromatography, the XL peptides are of relatively low abundance. To increase the percentage of crosslinked peptides, different enrichment methods (e.g., cation exchange) as well as the use of acidic crosslinkers (e.g., ADH) are recommended. After sorting the crosslinked peptides (scores ≤ 1 × 10-4_{), we ended up with 360 intra- and 79 inter-XL peptides. Redundant XLs linking}

residues from endogenous proteins (e.g., HDAC2, MBD2, and RBBP4, which were pulled down along with recombinant complex from EXPI293 cells) with the same crosslinked residues as recombinantly expressed proteins were eliminated from the data set. For example, QSQIQKEATAQK (90)-GKPDLNTALPVR (172) can be assigned between Lys90 in recombinantly expressed MBD3_Mouse and lysine (172) in P66A_Human or lysine90 in endogenously expressed MBD2_Human and lysine (172) in P66A_Human. After these filtering steps, a dataset of unique XLs comprising 22 inter- XLs and 90 intra XLs was obtained (Appendix E and F).

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Table 2. Percentage of XL types

Types Cross-linked Spectra Loop-linked Spectra Mono-linked Spectra Regular Spectra

Percent 12.9% 4.8% 13.9% 68.4%

Types Cross-linked peptides Loop-linked peptides Mono-linked Peptides Regular peptides

Percent 25.9% 7.3% 20.2% 46.5%

Next, to check the overall distribution of the XLs in the complex, the set of inter- and intra-XLs were visualized using the xiNET tool (Fig 4-14). As can be seen in Figure 4-14 the pattern of XLs indicates that the MBD domain of MBD3 lies adjacent to the MTA BAH, ELM and SANT domains and that GATAD2 shares XLs only with MBD3. The data also indicate that the N-terminal portion of HDAC1 contacts the N-terminal portion of MTA2. The number of detected inta-XLs was significantly higher in MBD3 than the other subunits.

Fig 4-14. Node-link diagram showing inter and intra XLs. Inter XLs obtained

for the MMH complex are shown with black lines. MBD3cc is a stabilized form of MBD3(1-291), which is fused with the interacting coiled-coil region of GATAD2A (residues 133–174).

The mass spectra corresponding to each XL were individually validated using the pLabel tool [223] to ensure that peptide sequences of the XLs match the corresponding spectra. Crosslinks with at least four fragment ions on both the alpha (α) and beta (β) chain were retained for modelling (in XL-MS/MS studies, the longer

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chain in crosslinked peptide is annotated as α chain and the shorter one is named β chain) [224]. It is notable that the number of XLs observed for each subunit is very different, which might be due to the secondary structure surrounding potential XL sites [173] or the number of crosslinkable residues. Figure 4-15 shows a fragmentation spectrum for a representative XL identified in this study.

Fig 4-15. Fragmentation spectrum of a representative crosslinked peptide identified in this study. Alpha peptide y- and b-ions are labelled in green and pink, respectively, while the beta peptide y- and b-ions are labelled in red and blue, respectively. The X axis is mass to charge ratio (m/z) and Y axis represents relative intensity.