Q exactive ms system

The SuperSESI source was coupled to the Q Exactive MS system (Thermo Fisher, Waltham, MA, USA) with the following instrumental parameters: sheath gas flow rate, 60; auxiliary gas flow rate, 2; spray voltage, 3.5 kV; capillary temperature, 275 °C; and S-lens RF level, 60.0. The MS was controlled directly using Q Exactive Tune software (version 2.9) in full MS mode. The scan range of negative ionization modes was set from 50 to 500 m/z; microscans of 1; AGC target of 1e6; and maximum injection time of 100 ms) with a high resolution of 140,000.

To identify the proteins interacting with CNR2, protein bands were displayed using silver-staining kit (Beyotime, Shanghai, China), Bands not detected in the IgG group were excised from the gels and performed LC–MS/MS analysis (Wininnovate Bio). Briefly, isolated protein samples were dried using a vacuum centrifuge, resuspended in 40 µl 50 mM ammonium bicarbonate, and prepared for LC–MS/MS. Tryptic peptide mixtures were separated using an Easy nLC UPLC system (Thermo Fisher, MA, USA) coupled with an in-house packed nanoViper C18 resin (3 µm, 100 Å) column (15 cm long 50 µm inner diameter). A 120-min gradient from 98% HPLC water/2% ACN/0.1% formic acid to 95% ACN/2% HPLC water/0.1% formic acid was used, and peptides were analyzed using a ThermoFisher Q Exactive MS system. Raw mass spectra were searched against a Uniprot Mouse Database and all MS/MS statistical analyses were performed utilizing PEAKS Studio 8.5 (version 8.5, Bioinformatics Solutions Inc. Waterloo, Canada) software.

PCR reagents, plasmid purification, restriction enzymes, and T4 DNA ligase were used as recommended by New England Biolabs (NEB) unless otherwise noted. Vectors used in this study included the bacterial expression vectors pCWori (43 (link)) and pET28a (Novagen). E. coli cell strains used in this study included Top10 (Invitrogen), DH5α, and BL21(DE3) (NEB). All reagents were used as received from commercial sources. Loganin (Cayman Chemical) and loganic acid (Arctom Chemicals) were 98+% pure.
LC-MS analyses of the Camptotheca SLAS mutants were conducted using a Shimadzu LC-MS2010EV system at the Institute for Genomic Biology, University of Illinois Urbana-Champaign. High resolution LC-MS analyses of the WT Camptotheca SLASs and the SLS, SLAS common ancestor were performed by Dr Alexander Ulanov in the Metabolomics Laboratory of the Roy. J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign using a Dionex Ultimate 3000 series HPLC system (Thermo Scientific) with Q-Exactive MS system (Thermo Scientific). Spectroscopic measurements were recorded with a Cary UV-Vis Bio100 dual beam spectrophotometer or Molecular Devices SpectraMax M-series plate reader as appropriate.

Basic protamines were independently isolated from wild type and transgenic cauda epididymal mouse sperm and ejaculated human sperm. All samples contained at least 10 million cells. Spermatozoa were lysed by hypotonic shock and the chromatin solubilized as described75 (link). The nucleoproteins were then extracted with HCl 0.5 N at 37 °C for 5 minutes and the precipitated with 20% trichloroacetic acid. Nuclear proteins were visualized in acid-urea polyacrylamide gels as described76 (link). Finally, intact nuclear proteins were detected by mass spectrometry using high performance liquid chromatography coupled with electrospray ionization and detection with the Q Exactive MS system (Thermo Fisher Scientific).