JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_1_WT_CMCEpeptide_timsHT_DDA_Slot2-12_1_6053.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_result.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_10_dYhbOdHchA_MGO_CMCEpeptide_timsHT_DDA_Slot2-15_1_6059.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_7_dYhbOdHchA_CMCEpeptide_timsHT_DDA_Slot2-14_1_6057.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_5_dYhbO_CMCEpeptide_timsHT_DDA_Slot2-18_1_6065.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_6_dYhbO_CMCEpeptide_timsHT_DDA_Slot2-21_1_6071.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_3_WT_CMCEpeptide_timsHT_DDA_Slot2-20_1_6069.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_2_WT_CMCEpeptide_timsHT_DDA_Slot2-17_1_6063.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_11_dYhbOdHchA_GO_CMCEpeptide_timsHT_DDA_Slot2-16_1_6061.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_4_dYhbO_CMCEpeptide_timsHT_DDA_Slot2-13_1_6055.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_8_dYhbOdHchA_CMCEpeptide_timsHT_DDA_Slot2-19_1_6067.d.ziphttps://storage.jpostdb.org/JPST004108/files/PJ1784_Watanabe_9_dYhbOdHchA_CMCEpeptide_timsHT_DDA_Slot2-22_1_6073.d.zipprimary200OKProteomicsHidetaka KosakoEscherichia Colihttps://repository.jpostdb.org/entry/JPST004108Tokushima UniversityjPOSTfalseLFQ of CML/CEL peptides from E. coli cellsEach bacterial strain was lysed in 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were solubilized by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 ºC. The supernatants were recovered, and proteins (1 mg each) were purified by methanol–chloroform precipitation and solubilized in 150 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After sonication and heating, the protein solutions were digested with 10 µg of trypsin/Lys-C mix (Promega) at 37 ºC overnight. The resulting peptide solutions were diluted 6-fold with HBS (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl), centrifuged, and subjected to immunoaffinity enrichment using the PTMScan Carboxymethyl/Carboxyethyl Lysine Motif kit (Cell Signaling Technology). The eluates in 0.15% TFA and 5% acetonitrile were desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and re-dissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 µL/min starting at 3% B, which was linearly ramped to 32% B over 90 min, then raised to 95% B at 91 min, and held at that level until 101 min. The mass spectrometer was operated in parallel accumulation–serial fragmentation (PASEF) mode. The m/z range for both MS1 and MS2 spectra was 100-1700, and the ion mobility range was 0.6-1.6 V·s/cm2. The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied in the m/z and ion mobility space to exclude low m/z, singly charged ions from precursor selection. The raw data were processed using the FragPipe (v22.0). Database searches were performed with the MSFragger (v4.1), employing the default parameters of the LFQ workflow against the UniProt E. coli (strain K12) database (6,472 entries). Carbamidomethylation of cysteine (+57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of protein N-terminus (+42.0106 Da); oxidation of methionine (+15.9949 Da); carboxymethylation (+58.0055 Da) or carboxyethylation (+72.0211 Da) of lysine. The resulting identifications were filtered using Philosopher with default parameters (MSBooster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings.Mon Jun 22 00:00:00 BST 2026PXD069131562