Apollo 2 esi source

Intact-protein mass analysis was performed on a 1290 Infinity UHPLC (Agilent Technologies, Waldbronn, Germany), coupled to an Impact II QTOF-MS with Apollo II ESI source (Bruker Daltonics, Bremen, Germany). Reversed-phase separation was performed on a PLRP-S column (Agilent Technologies), with 5 µm particle size, 1000 Å pore size, 1 mm inner diameter, and 50 mm length. Mass spectra were recorded in positive ion ESI mode and the mass range from 600 to 7000 m/z (Details of LC-MS method in Supplementary Material). Data were analyzed in a Compass Data analysis 4.4 (Bruker Daltonics) tool. A weighted average of DEPC label incorporation was calculated for each sample. The weighted average of labels was calculated using the following formula:
$$\mathrm{Average} \mathrm{label} \mathrm{number}=\frac{\sum _{\mathrm{x}=1}^{\mathrm{n}}\mathrm{x}{\mathrm{I}}_{\mathrm{x}}^{\mathrm{M}\mathrm{o}\mathrm{d}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{e}\mathrm{d}}}{\sum _{\mathrm{x}=1}^{\mathrm{n}}{\mathrm{I}}_{\mathrm{x}}^{\mathrm{M}\mathrm{o}\mathrm{d}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{e}\mathrm{d}}+{\mathrm{I}}_0^{\mathrm{U}\mathrm{n}\mathrm{m}\mathrm{o}\mathrm{d}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{e}\mathrm{d}}}$$ where x is the number of DEPC modifications, I₀ is the intensity of unlabeled peaks, and I_x is the intensity of labeled peaks [35 (link)].

UHPLC-Q-TOF-MS analyses were performed on a Waters Acquity UHPLC System (Waters, Eschborn, Germany) coupled to a Bruker maXis UHR-ToF-MS with an Apollo II ESI source (Bruker Daltonics, Bremen, Germany). A YMC Triart C18 column (150 × 3.0 mm, 3 μm, YMC, Dinslaken, Germany) was used for separation at 30°C. 10 mM ammonium formate buffer (pH = 4) and methanol were used as mobile phases A and B, respectively. The detailed operating parameters are shown in the Supplementary Material.

All synthesized and purified reference compounds were characterized based on exact mass and fragmentation spectra, which are shown in Online Resource 1. An HPLC-qToF-MS coupling was used including an Elute pump (Bruker Daltonics, Bremen, Germany), a column oven (Bruker Daltonics) set to 40 °C, and a PAL HTC-xt autosampler (CTC Analytics, Zwingen, Switzerland) cooled to 8 °C. Furthermore, an Impact II with an Apollo II ESI source (Bruker Daltonics) was selected for mass spectrometric detection. The chromatographic separation was carried out on a Nucleodur C18 Pyramid column (100 × 2 mm, 3 µm, Macherey–Nagel) with an ACN/water gradient supplemented with 0.1% FA. The detailed gradient, further mass spectrometric parameters, and fragmentation data are shown in Online Resource 1 (Tables S9 and S10, Figures S1-S5). The same instrument setup and separation method coupled to a DAD (Bruker Daltonics) were used for the purity check.