Phosphopeptides were separated by liquid chromatography on the EASY-nLC 1200 system (Thermo Fisher Scientific) for 120 minutes. Briefly, phosphopeptides were loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100; 100 μm × 2 cm, nanoViper C18) connected to a C18-reversed phase analytical column (Thermo Scientific Easy Column; 10-cm long, 75-μm inner diameter, 3 μm resin). The phosphopeptides were separated by reversed-phase chromatography using a binary buffer system consisting of 0.1% formic acid (buffer A) and 80% acetonitrile in 0.1% formic acid (buffer B) at a flow rate of 300 nl/minute. MS data were analyzed on a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) using a data-dependent top 10 method, with a maximum injection time of 10 ms, scan range of 300–1800 m/z and automatic gain control target of 3e6. Survey scans were acquired at a resolution of 70 000, and the resolution for HCD spectra was set to 17 500. Normalized collision energy was 30 eV, and the underfill ratio was set to 0.1%.
MS data were processed with Proteome Discoverer version 2.4. Enzyme specificity was set to that of trypsin, allowing for cleavage up to two missed cleavage sites. Carbamidomethyl (C), TMT 6/10 plex (N-term) and TMT 6/10/16 plex (K) were selected as fixed modifications, while Oxidation (M), TMT 6/10/16 plex (Y) and phospho (S/T/Y) was added as variable modifications. The FDR for phosphopeptides was set to 0.01. Searches were performed against the Gallus gallus UniProt FASTA database (February 2020) containing 34 878 entries. Quantification of phosphopeptides was normalized by subtracting the median intensity of each sample. Phosphopeptides that changed by > 1.5 fold with a Student t-test p < 0.05 were considered significantly regulated.
With regard to the kinase–substrate enrichment analysis, iGPS [58 (link)] was used to identify any phosphosite on a kinase. GSEA version 4.1.0 [59 (link)] was used to identify significantly enriched kinases using a ranked fold change of all quantified phosphopeptides. The corresponding p value and normalized enrichment score were assigned for each kinase.
MS data were processed with Proteome Discoverer version 2.4. Enzyme specificity was set to that of trypsin, allowing for cleavage up to two missed cleavage sites. Carbamidomethyl (C), TMT 6/10 plex (N-term) and TMT 6/10/16 plex (K) were selected as fixed modifications, while Oxidation (M), TMT 6/10/16 plex (Y) and phospho (S/T/Y) was added as variable modifications. The FDR for phosphopeptides was set to 0.01. Searches were performed against the Gallus gallus UniProt FASTA database (February 2020) containing 34 878 entries. Quantification of phosphopeptides was normalized by subtracting the median intensity of each sample. Phosphopeptides that changed by > 1.5 fold with a Student t-test p < 0.05 were considered significantly regulated.
With regard to the kinase–substrate enrichment analysis, iGPS [58 (link)] was used to identify any phosphosite on a kinase. GSEA version 4.1.0 [59 (link)] was used to identify significantly enriched kinases using a ranked fold change of all quantified phosphopeptides. The corresponding p value and normalized enrichment score were assigned for each kinase.
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