C18 oasis cartridges

TPC was determined following a method by Georgé et al. (2005) [39 (link)]. The extraction was made using a solution of 70% acetone (v/v) under magnetic stirring for 45 min, using 0.3–1.0 g of freeze-dried samples. The mixture was centrifuged for 10 min at 3500 rpm and the supernatant raw extract was recovered in Eppendorf vials. TPC A non-soluble fraction and the soluble compound TPC B fraction were evaluated from the raw extract. For the A fraction, a series of solutions of 25, 50 and 75 µL of the raw extract in 500 mL of pure methanol were prepared. The separation of soluble compounds was done by solid phase extraction (SPE) using C18 OASIS cartridges (Waters Corp.; Milford, MA, USA) previously conditioned following the fabricant instructions. 500 µL of raw extract were diluted in 3.5 mL of distilled water and a 2 mL aliquot of this solution was injected into the OASIS cartridge.
The quantification of A and B fractions was done using the Folin-Ciocalteu method with a UV-VIS spectrophotometer Shimadzu 2200 (Kioto, Japan) at 760 nm and both fractions were used (Abs_B-Abs_A) to corrected interferences. Gallic acid was used as standard. The curve was stablished (y = 0.0011x + 0.0529, R² = 0.9997). The total polyphenol content was expressed in terms of mg of gallic acid equivalents GAE/100 g of the DW sample.

For each treatment condition, five independent biological replicates were performed. Cells were washed twice with cold phosphate-buffered saline supplemented with 1 mM Na₃VO₄ and 1 mM NaF, and lysed in 0.5 mL of urea buffer [8 M urea in 20 mM HEPES (pH 8.0), supplemented with 1 mM Na₃VO₄, 1 mM NaF, 1 mM Na₄P₂O₇ and 1 mM β-glycerophosphate]. Cell lysates were further homogenised by sonication (three cycles of 10 s on and 10 s off) and insoluble material was removed by centrifugation. Protein was quantified by the BCI assay. For each replicate, 325 μg of protein was reduced, alkylated and digested with TLCK-trypsin (Thermo Fisher Scientific) as previously described.^{15 (link)} The resultant peptide solutions were desalted with C18-Oasis cartridges (Waters, Manchester, UK) as indicated by the manufacturer with slight modifications as previously described.^{16 (link)} Enrichment of phosphorylated peptides was performed with TiO₂ as previously described.^{15 (link),16 (link)}