Sep pak c18 1 cc vac cartridges

Methanol was added to 100 μg of the pooled supernatants to a final concentration of 70% and further processed according to the R2P1 protocol (47 ), with tryptic digestion performed in 50 mM Hepes (pH 8.5). Elution fractions were combined and dried by vacuum centrifugation. Dried samples were resuspended in deionized water, and TMT labeling was performed according to Zecha et al. (35 (link)). All 15 samples (5 treatment conditions in triplicates) were labeled with one TMTpro plex. Next, equal amounts were pooled and desalted by C18 solid-phase extraction cartridges (Sep-Pak Vac 1 cc C18 Cartridges, Waters Corp). Desalted peptides were chromatographically separated via basic reversed phase chromatography as previously described (36 (link)) into 24 fractions, dried down, and stored at −20 °C until further analysis.

The analytical method was revised in accordance with other protocols found in the literature [23 (link),24 (link)]. As a first step, the internal standard (IS) fludrocortisone (10 ng) was added to the samples from treated mice, and Dex (0–300 ng) and fludrocortisone (10 ng) was included for the calibration curve. The liver, brain, and spinal cord tissue samples were treated with methanol (1:4 w/v) and acetonitrile (1:1 w/v), stirred, and sonicated for 20′. Then, water (1:10 w/v) was added, and the stirring and sonication steps were repeated. The resulting samples were centrifuged at 7000× g for 15 min at 4 °C. The supernatant was further purified with solid-phase extraction using Sep-Pak C18 1 cc Vac Cartridges (Waters, Milford, MA, USA) and was conditioned before use with 1 mL methanol, followed by 1 mL water. Samples were loaded on the SPE columns and passed through dropwise. Finally, the cartridges were rinsed with 1 mL water:acetone (80:20) and then with 1 mL of water before drying the columns under a vacuum for 5′. Samples were eluted with 1.8 mL acetonitrile into glass receiving tubes. Plasma aliquots were directly eluted with acetonitrile (1:4 v/v) and centrifuged at 7000× g for 15 min at 4 °C. All samples were evaporated to remove the organic phase. Just before analysis, they were suspended in 100 μL of 0.05% acetic acid:acetonitrile (80:20).

The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, IL); hydrazide support and sodium periodate were purchased from Bio-Rad (Hercules, CA); sequencing-grade trypsin was purchased from Promega (Madison, WI); PNGase F was purchased from New England Biolabs (Ipswich, MA); Sep-Pak C18 1 cc Vac Cartridges were purchased from Waters (Milford, MA); and acetonitrile (ACN), trifluoroacetic acid (TFA), formic acid, urea, tris(2-carboxyethyl)phosphine (TCEP), iodoacetamide, phorbol 12-myristate 13-acetate (PMA), 5 M NaCl, sodium dodecyl sulfate (SDS), 10× phosphate buffered saline buffer (PBS) pH 7.4 and 1 M Tris–HCl pH 8.0 buffer were purchased from Sigma-Aldrich (St. Louis, MO).

All chemicals used were of HPLC-grade purity. Unless otherwise specified, all chemicals and reagents were obtained from Sigma-Aldrich (Steinheim, Germany). Formic acid (FA) was from Merck (Darmstadt, Germany). Acetonitrile (ACN), chloroform (CHCl₃), and methanol (MeOH) were purchased from Biosolve (Valkenswaard, The Netherlands). The Sep-Pak C18 1cc vac cartridges were purchased from Waters (MA, USA). MilliQ was produced by an in-house system (Millipore, Billerica, MA).

Peptides were desalted with Sep-Pak C18 1 cc Vac Cartridges (Waters) over a vacuum manifold. Tryptic digests were first acidified by addition of 16.6 μL trifluoroacetic acid (TFA, Acros) to a final concentration of 1% (vol/vol). Cartridges were first conditioned (1 mL 80% ACN, 0.5% TFA) and equilibrated (4 × 1 mL 0.5% TFA) before loading the sample slowly under a diminished vacuum (ca. 1 mL/min). The columns were then washed (4 × 1 mL 0.5% TFA), and peptides were eluted by addition of 1 mL elution buffer (80% ACN, 0.5% TFA). During elution, vacuum cartridges were suspended above 15 mL conical tubes, placed in a swing-bucket rotor (Eppendorf 5910 R), and spun for 3 min at 350 × g. Eluted peptides were transferred from Falcon tubes back into microfuge tubes and dried using a vacuum centrifuge (Eppendorf Vacufuge). Dried peptides were stored at −80 °C until analysis. For analysis, samples were vigorously resuspended in 0.1% FA in Optima water (ThermoFisher) to a final concentration of 0.5 mg mL⁻¹.