Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD063886/PXD063886_community_annotated.sdrf.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD063886/4916-qiege.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD063886/5053-HXSA1-C.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD063886/4916-qiege.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD063886/5053-HXSA1-C.rawprimary200OK1912548105@qq.comMengling LiMass SpectrometryBottom-up proteomicshttps://www.ebi.ac.uk/pride/archive/projects/PXD063886Cell CultureThe cleaved HxsA protein was analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Briefly, the E. coli BL21(DE3) cells containing the HXS system was cultured overnight at 37°C and collected by centrifugation at 6,708 g for 10 min. The pellets were resuspended in SDS-PAGE loading buffer, and the cleaved HxsA protein was separated on an SDS-PAGE gel and confirmed by western blottingusing HxsA-specific antibodies. The band corresponding to cleaved HxsA on the second SDS-PAGE gel was carefully excised, and the HxsA proteins were recovered using electrophoresis. The sample was sent to the Sangon Biotech (Shanghai, China) for LC-MS/MS analysis. The recovered HxsA protein was divided into two aliquots and subjected to parallel in-solution digestion using either trypsin or chymotrypsin.Both digests were analyzed independently by LC-MS/MS. MS data were acquired on a Q Exactive Plus mass spectrometer (Thermo scientific)coupled to an EASY nLC 1200 system (Thermo scientific). Briefly, samples were first loaded onto a nanoViper C18 trap column (3 μm, Thermo Scientific) for on-line desalting. After washing the trap column with loading solvent to remove salts, peptideswere eluted from the trap and separated on an analytical C18 column (Acclaim PepMap RSLC, 75 μm × 25 cm, C18, 2 μm, 100 Å; Thermo Scientific) using solvent A (0.1% formic acid in water) and solvent B (80% acetonitrile, 0.1% formic acid). Peptide separation was performed at a flow rate of 0.300 μL/min with the following gradient: 5% B at 5.0 min, increased to 40% B at 12.0 min, ramped to 95% B at 14.0 min, and held at 95% B until 20.0 min for column washing. The system was then returned to 5% B at 20.1 min and re-equilibrated prior to the next injection. The total method time was 25 min. MS full scans were performed in the Orbitrap mass analyzer in the m/z range of 300-1500 with a resolution of 70,000, an automatic gain control (AGC) set at 3e6 and with a maximum injection time of 100 ms. The 20 most intense ions were isolated and further fragmented via high-energy collision dissociation (HCD). MS/MS (MS2) spectra were acquired in the Orbitrap over an m/z range of 200-2000 at a resolution of 17,500, with an AGC set at 1e5 and with a maximum injection time of 50 ms. We selected ions with charge state from 2+ to 5+ for screening. Normalized collision energy (NCE) was set at 28 and 30 and a dynamic exclusion of 25 s.PrideNot availableRaw MS data were processed using Proteome Discoverer (version 2.5, Thermo Fisher Scientific) and searched against the HxsA protein sequence.Carbamidomethylation of cysteine residues (+57.021 Da) was set as a static modification, whereas oxidation of methionine and tyrosine (+15.995 Da), as well as HxsB/C-catalyzed modifications (+12.000 Da or -2.016 Da) on histidine, serine and arginine residues, were set as dynamic modifications. The precursor ion mass tolerance was set to 10 ppm and the fragment ion mass tolerance to 0.02 Da. Peptide-spectrummatches were filtered using a false discovery rate of 1% at the peptide level. Modification site assignment was confirmed by manual inspection of fragment ionspectra.ProteomicsMengling LiHuazhong Agricultural UniversityEscherichia ColiPARTIAL41826297 Li M, Sun E, Wang S, Huang Y, Song H, Kuang S, Li G, Liu Y, Xia Z, Zhang X, Han J, Dong Z, Rao VB, Sun L, Tao P. Periplasmic gatekeeping of phage DNA entry by an rSAM enzyme matured effector with HxS repeats. Nat Commun. 2026 17(1):3910 10.1038/s41467-026-70567-01912548105@qq.comHuazhong Agricultural UniversityChinaIn the relentless arms race between bacteria and phages, bacteria have evolved a variety of defense strategies to combat phage infection. However, no system has been previously demonstrated to specifically inhibit phage genomic DNA ejection. Here, we present a bacterial antiphage system, termed HXS, which provides broad-spectrum and robust antiphage activity by interfering with phage DNA entry. The HXS system consists of two radical S-adenosylmethionine (rSAM) enzymes (HxsB and HxsC), a small protein (HxsD), and the effector HxsA with a peptidoglycan-binding domain and five His-Xaa-Ser (HXS) repeats. HxsB/HxsC catalyze rSAM enzyme-dependent maturation of HxsA, including N-terminal processing and a site-specific +8 Da modification, thereby producing a periplasmic effector required for HXS defense. Biochemical evidence supports a model in which the matured effector likely engages incoming DNA electrostatically to arrest entry, establishing an rSAM enzyme-modified protein effector in antiphage defense.Periplasmic gatekeeping of phage DNA entry by an rSAM enzyme matured effector with HxS repeats.Li Mengling M, Sun Erchao E, Wang Shuangshuang S, Huang Yuxuan Y, Song Haiguang H, Kuang Sirong S, Li Guoqi G, Liu Yuepeng Y, Xia Ziwei Z, Zhang Xueqi X, Han Jiumin J, Dong Zhaoxu Z, Rao Venigalla B VB, Sun Lvhui L, Tao Pan PfalseThe cleaved form of HxsA protein derived from the E. coli HXS systemIdentification of the cleaved form of HxsA protein derived from the E. coli HXS 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