Proteins were digested as described recently for the mouse retina (Sze et al., 2021 (link)). In brief, a total of 50 μg proteins were reduced with DTT for 10 min, then alkylated with IAA at room temperature for 10 min with protection from light. The alkylation reaction was quenched by adding 0.2% phosphoric acid. Samples were added to the S-Trap protein binding buffer, and proteins were trapped by a filter in the S-Trap Micro Spin Column (Protifi, United States) (HaileMariam et al., 2018 (link)). After trypsin (Promega, United States) digestion, the peptides were resuspended with 0.1% FA for LC-MS/MS analysis (calibrated at 0.5 μg/μl) using Pierce Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, United States).
Both DDA and SWATH-MS analyses were performed by the TripleTOF 6,600 system (SCIEX, MA, United States) connected to an Eksigent ekspert™ nanoLC415 system similar to our previous protocols (Shan et al., 2018b (link); Cheung et al., 2020 (link); Bian et al., 2021 (link)). For either IDA or SWATH acquisitions, 2 µg peptide was loaded to a trap column (100 μm × 2 cm, C18) for 15 min. Then, it was separated on a nano-LC column (100 μm × 30 cm, C18, 5 µm). An isolation of 100 Variable windows was selected in a looped mode over the full mass range of 100–1800 m/z scan in SWATH acquisition.
Both DDA and SWATH-MS analyses were performed by the TripleTOF 6,600 system (SCIEX, MA, United States) connected to an Eksigent ekspert™ nanoLC415 system similar to our previous protocols (Shan et al., 2018b (link); Cheung et al., 2020 (link); Bian et al., 2021 (link)). For either IDA or SWATH acquisitions, 2 µg peptide was loaded to a trap column (100 μm × 2 cm, C18) for 15 min. Then, it was separated on a nano-LC column (100 μm × 30 cm, C18, 5 µm). An isolation of 100 Variable windows was selected in a looped mode over the full mass range of 100–1800 m/z scan in SWATH acquisition.
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