Nano orbitrap fusion lumos tribrid mass spectrometer system

The identification of degraded SNAP43 fragment (the band labeled as asterisk in Supplementary Fig. 1a) was carried out by liquid chromatography-mass spectrometry. Briefly, peptides prepared from in-solution digestion were analyzed by nano system (Thermo Scientific, EASY-nLC1200) coupled with a 1,000,000 FWHM high-resolution Nano Orbitrap Fusion Lumos Tribrid Mass Spectrometer system (Thermo Scientific). The raw files from Orbitrap Lumos were imported into Proteome Discoverer software 2.3 (Thermo Scientific) with the Sequest HT search engine against proteome sequence for data processing. Finally, the target band was identified as the carboxyl terminal degraded SNAP43, with 47.55% coverage (source data are provided as a Source Data file).

The purified LdhA cultured in Luria–Bertani (LB) with 2% glucose was detected by 12% SDS-PAGE, the excised LdhA band was digested with trypsin by a standard in-gel digestion protocol. The trypsin digested product was desalted by Ziptip C18 chromatographic column and redissolved by 0.1% trifluoroacetic acid. Peptides were separated on a nanoViper C18 silica column Acclaim PepMap RSLC (75 μm × 25 cm, 2 μm, Thermo, United States) with mobile phase system of solvent A (0.1% formic acid) and B (80% ACN and 0.1% formic acid) at a flow rate of 300 nL/min, and analyzed by nano system (Thermo Scientific, EASY-nLC, United States) coupled with a 1,000,000 FWHM high-resolution Nano Orbitrap Fusion Lumos Tribrid Mass Spectrometer system (Thermo Scientific, United States). Mass spectrometric data processing used the Proteome Discoverer software 2.3 (Thermo Scientific, United States).