Fig 2: Mass spectrometric analysis of PRDM9–DNA complex.(A) To analyse the PRDM9–DNA complex with mass spectrometry, 7 µl of Halo-ZnFCst 1–11 was incubated with a 2 µM 75-bp DNA of the Hlx1 hotspot for 60 min at room temperature, separated on a native 5% polyacrylamide gel, and visualized by Coomassie blue staining (arrow). As a control, the protein without DNA was loaded on the gel not giving a distinct band under native conditions. “Plus” and “minus” indicate the presence or absence of PRDM9 and DNA within the binding reaction. (B) Shown is the mass spectrum of the PRDM9–DNA complex (Halo-ZnFCst 1–11 + single-Hlx1 75 bp), which was cut out and isolated of the native gel shown in (A), treated with trypsin digestion, DTT reduction and carbamethylation using iodacetamide, and finally analysed using a MALDI-TOF Axima Performance instrument (Shimadzu). The spectrum shows the PMF of the PRDM9–DNA complex with all prominent peaks matching the peptides of our protein. (C) To confirm the PMF data, four colour-coded MS/MS spectra of prominent m/z values were obtained: 1,338.61 (blue), 1,767.84 (purple), 1,810.76 (green), and 1,908.01 (orange). Note that the raw data and m/z values can be found in Tables S6 and S7.

Fig 3: MALDI-TOF MS analysis of intact LOS indicates that the transposon mutants make truncated LOS compared to wild type. LOSs from B. thetaiotaomicron ?CPS, B. thetaiotaomicron tnBT3365, B. thetaiotaomicron tnBT3368, and B. thetaiotaomicron tnBT3376 strains were isolated using the large-scale LPS/LOS extraction method. The resulting material was desalted and spotted on a THAP-nitrocellulose matrix for analysis on a Shimadzu Axima Performance MALDI-TOF mass spectrometer in linear negative-ion mode. LOS peaks of interest have been colored, with each color representing a different LOS species present in the different strains. Peaks colored gray are likely derived from capsular polysaccharide (those that do not appear in the B. thetaiotaomicron ?CPS spectrum) or lipid A in the case of the peak clusters around 1,650 to 1,700 m/z.