Tf 20 l r s

From the same peptide digest above, ˜95% of peptides per sample was used for phosphoproteomic analysis. The phosphopeptide enrichment was performed using the High-Select^™ Fe-NTA kit (Thermo Scientific, A32992) according to the kit instruction, as described previously [104 (link)]. Briefly, the resins of one spin column in the kit were divided into five equal aliquots, each used for one sample. The peptide-resin mixture was incubated for 30 min at room temperature and then transferred into the filter tip (TF-20-L-R-S, Axygen). The supernatant was removed after centrifugation. Then the resins adsorbed with phosphopeptides were washed sequentially with 200 µL× 3 washing buffer (80% ACN, 0.1% TFA) and 200 µL×3 H2O to remove nonspecifically adsorbed peptides. The phosphopeptides were eluted off the resins by 100 µL×2 elution buffer (50% ACN, 5% NH3 H2O). All centrifugation steps above were conducted at 500 g, 30 sec. The eluates were collected for speed-vac and dried for mass spectrometry analysis.

Besides ~4 μg of peptides digested per sample that were used for proteomic analysis, all the peptides from different replicates of equal amount were pooled for phosphopeptide enrichment and phosphoproteomics, due to the limited peptide amounts yielded in individual replicate. The phosphopeptide enrichment was performed using the High-Select Fe-NTA kit (Thermo Fisher Scientific, A32992) according to the manufacturer’s instructions (68 (link)). Briefly, the resins of spin-column in the Fe-NTA kit were aliquoted and incubated with 80 to 300 μg of total peptides for 30 min at room temperature. The resins were then transferred into a filter tip (TF-20-L-R-S, Axygen), so that the supernatant was removed by centrifugation. Then, the resins were washed sequentially with 200 μl of washing buffer (80% acetonitrile (ACN) and 0.1% trifluoroacetic acid) three times and 200 μl of liquid chromatography (LC)–MS grade H₂O two times to remove the nonspecifically adsorbed peptides. The enriched phosphopeptides were then eluted off the resins by 100 μl of elution buffer (50% ACN and 5% NH₃·H₂O) two times. All the centrifugation steps were kept at 500g, 30 s. The eluates per species were combined and dried by speed-vac and stored in −80°C.

The phosphopeptide enrichment was performed using the High-Select Fe-NTA kit (Thermo Fisher Scientific, A32992) according to the manufacturer’s instructions. Briefly, the resins of the spin column were aliquoted, incubated with 200 μg of total peptides for 30 min at room temperature, and transferred into the filter tip (TF-20-L-R-S, Axygen). The supernatant was then removed by centrifugation. Then, the resins adsorbed with phosphopeptides were washed three times with 200 μl of washing buffer (80% ACN and 0.1% trifluoroacetic acid) and twice 200 μl of water to remove nonspecifically adsorbed peptides. The phosphopeptides were then eluted off the resins twice with 100 μl of elution buffer (50% ACN and 5% NH₃•H₂O). The centrifugation steps above were all kept at 500g for 30 s. The eluates were dried by SpeedVac and stored at −80°C before MS measurements.