Ekspert nanolc 415 system

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.

All fractions were evaluated by using a 5600 Triple-TOF mass spectrometer that was directly linked to a reversed-phase high-pressure liquid chromatography Ekspert-nanoLC 415 system (Eksigent, Dublin, CA). Formic acid (0.1%) in water was used as mobile phase A, and mobile phase B was 0.1% formic acid in acetonitrile. All fractions were eluted from the analytical column at a flow rate of 250 nl/min using an initial gradient elution of 10% B from 0 to 5 min, transitioned to 40% over 120 min, ramping up to 90% B for 5 min, holding 90% B for 10 min, followed by reequilibration of 5% B at 10 min with a total run time of 150 min. Peptides were injected into the mass spectrometer using a 10-μm SilicaTip electrospray PicoTip emitter. Mass spectra (MS) and tandem mass spectra (MS/MS) were recorded in positive-ion and high-sensitivity mode with a resolution of ∼35,000 full-width half-maximum. Before testing samples on the mass spectrometer, calibration of spectra occurred after the acquisition of every sample using dynamic LC-MS and MS/MS acquisitions of 100 fmol of β-galactosidase. The ion accumulation time was set to 250 ms (MS) or 70 ms (MS/MS). The collected raw files spectra were stored in .wiff format.

Retention time calibration peptides (iRT-Kit, Biognosys, Schlieren, Switzerland) were spiked into each sample at 75× dilution of the iRT stock solution. SWATH–MS was carried out on a 5600+ triple-TOF mass spectrometer coupled to an Ekspert NanoLC 415 system (Eksigent, AB Sciex, Dublin, CA, USA) equipped with an in-house packed emitter tip column filled with 2.6 µm Aeris C18 material (Phenomenex, Torrance, CA, USA) on a length of 20 cm. Peptides were separated over a 120-minute gradient using a binary solvent system (solvent A: 1% ACN, 0.1% FA in water, solvent B: 90% ACN, 0.1% FA in water). To generate spectral libraries, the mass spectrometer was run in data-dependent acquisition (DDA) mode (technical details in Supplementary Methods). SWATH–MS spectra were acquired over a 2-hour gradient using variable window width for precursor ion selection (technical details in Supplementary Methods). Each sample was injected three times, and the median ion intensity was calculated across the three SWATH–MS runs.