High pH reversed-phase liquid chromatography separation
Samples were fractionated to increase the proteomic depth, using high pH reverse phase separation. The peptides resuspended in buffer A (2% acetonitrile, containing ammonium hydroxide solution, at pH 10) were separated by ACQUITY UPLC (Waters, Milford, MA, USA) connected to a high pH UPLC column. Gradient elution was achieved at a rate of 200 μl min-1 for 66 min with buffer B (80% acetonitrile, containing ammonium hydroxide solution, at pH 10). Twenty fractions were collected from each sample and were pooled into 10 total fractions.
Liquid chromatography-mass spectrometry (LC-MS/MS) analysis
Mass spectrometry analysis was performed on a Q Exactive mass spectrometer coupled with Easy-nLC 1200 (ThermoFisher Scientific). The 2 μg peptides loaded onto the C18 column in buffer A (2% acetonitrile, 0.1% formic acid) were separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl min-1 in Easy-nLC 1200. The eluted peptides were injected into a Q Exactive mass spectrometer (ThermoFisher Scientific), using 1.8 kV electrospray voltage. The acquisition of survey full-scan MS spectra (m/z 350-1300) was attributed to a mass resolution of 70,000, followed by 20 sequential high energy collisional dissociation MS/MS scans at a resolution of 35,000.
For protein identification, MS/MS spectra were searched using Protein DiscovererTMSoftware 2.2 and parent proteins were identified by the highest score for a given peptide mass. The following settings were used: a maximum of two miscleavages; carbamidomethylation of cysteines as fixed modification; protein N-terminal acetylation and oxidation of methionines as variable modifications. At least one unique peptide containing a minimum of six amino acids sequence was required for per protein. The false discovery rate (FDR) was set to 1% to validate peptide spectra.