6530 accurate mass q tof instrument

The putative OmpK35, OmpK36, and empty gel bands (at the height of band 10) were excised from the SDS-PAGE gel for peptide analysis. The analysis was performed as previously described [17 (link)] by the Yonsei Proteome Research Center, Seoul, South Korea. A nano high performance liquid chromatography system (Agilent, Santa Clara, CA, USA) was used for nano LC-MS/MS analysis. Peptide separation was carried out using a nano chip column. Product ion spectra were analyzed using an Agilent 6530 Accurate-Mass Q-TOF instrument.
The MASCOT algorithm (Matrix Science, London, UK) was used for database searching to identify protein sequences. The criteria used were, taxonomy; Proteobacteria (NCBInr downloaded 2015.01.23), OmpK35 (accession no. ADG27468), OmpK36 (accession no. YP_005228001); fixed modification: carbamidomethylated at cysteine residues; variable modification: oxidized at methionine residues; maximum allowed missed cleavage: 2; MS tolerance: 100 ppm; MS/MS tolerance: 0.1 Da. Only peptides obtained from trypsin digestion were considered.

UV spectra were recorded on the Shimadzu UV-Vis Spectrophotometer, UV-1700 (Kyoto, Japan). IR spectra (neat) were recorded on the Perkin-Elmer FT-IR Spectrometer, SPECTRUM 2000 (Waltham, MA, USA). NMR spectra were acquired on a Bruker Avance III spectrometer (Billerica, MA, USA) operating at 600 MHz for ¹H and 150 MHz for ¹³C and equipped with a 3 mm cryogenically cooled probe. HRESIMS data were acquired on an Agilent 6530 Accurate Mass Q-TOF instrument (Santa Clara, CA, USA). Initial purification of the dichloromethane extract was carried out on a Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) column. Further purification of column fractions was performed using silica gel F254 PLC plates (1 mm thickness) (Merck KGaA, Darmstadt, Germany).