Thermo scientific ltq orbitrap xl mass spectrometer

The abundance of RbsB wild-type and mutants in the E. coli periplasmic space was analyzed using direct peptide mass identification, as described previously^{12 (link)}. Periplasmic fraction was prepared by EDTA–ice treatment^{12 (link)} from E. coli BW25113 ∆rbsB carrying pSYK1 and the pSTVP_AA-rbsB derivatives (Table 2). Periplasmic protein fractions were separated by SDS-PAGE and proteins in the size range between 28 and 36 kDa were excised from the gel. Proteins were analyzed by the UNIL Proteome Facility (https://www.unil.ch/paf/en/home.html). In short: samples were digested with trypsin and peptides were separated on an Ultimate 3000 Nano LC System (Dionex), followed by detection in a Thermo Scientific LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Mass spectra were analyzed using Scaffold Viewer 4, using thresholds of 99.9%. The minimum number of peptides for identification was 2.

Optical rotations were measured with a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). UV spectra were taken on a Beckman DU640 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA). IR spectra were recorded on a NICOLET NEXUS 470 spectrophotometer (International Equipment Trading Ltd., Vernon Hills, IL, USA) in KBr discs, with υ in cm⁻¹. ¹H-, ¹³C-NMR, DEPT, and 2D NMR spectra were recorded on a JEOL JNM-ECP 600 (JEOL Ltd., Tokoyo, Japan) and Varian 500 spectrophotometers (Varian, Palo Alto, CA, USA), with δ in ppm with solvent residual signals as internal standards (DMSO: δ_H 2.50 ppm, δ_C 39.5 ppm), and J in Hz. Semi-preparative HPLC was performed using an ODS column (YMC-Pack ODS-A, 10 × 250 mm, 5 µm) (YMC Co. Ltd., Kyoto, Japan).; The ratios of solvent were described as a mixture by v/v. HR-ESI-MS was measured on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) in m/z. A Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and silica gel (SiO₂; 100–200 mesh, 200–300 mesh, and 300–400 mesh; Qingdao Marine Chemical Inc., Qingdao, China) were used for column chromatography (CC), and TLC was carried out on silica gel GF₂₅₄ (10–40 mm; Qingdao Marine Chemical Inc., Qingdao, China) plates; spots were visualized under UV light and by spraying with 5% H₂SO₄ in C₂H₅OH (v/v) followed by heating.

UV spectra were recorded on Beckman DU 640 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA). IR spectra were taken on Bruker tensor-27 spectrophotometer in KBr discs (Bruker Corporation, Billerica, MA, USA). Specific rotations were measured on JASCO P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan). ESIMS were obtained on Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or Micromass Q-TOF ULTIMA GLOBAL GAA076 LC Mass spectrometer (Wasters Corporation, Milford, MA, USA). CD spectra were measured on JASCO J-715 spectropolarimeter (JASCO Corporation, Tokyo, Japan). NMR spectra were recorded on Agilent 500 MHz DD2 spectrometer using TMS as internal standard and chemical shifts were recorded as δ-values (Agilent Technologies Inc., Santa Clara, CA, USA). Semi-preparative HPLC was performed on an ODS column (HPLC (YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 3 mL/min)) (YMC Co., Ltd., Kyoto, Japan). Medium-pressure preparation liquid chromatography (MPLC) was performed on a Bona-Agela CHEETAHTM HP100 (Beijing Agela Technologies Co., Ltd., Beijing, China). Column chromatography (CC) were performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), and Sephadex LH-20 (Amersham Biosciences, San Francisco, CA, USA), respectively [27 (link)].