LC–MS/MS Analysis and Data Processing

For total peptide analysis, dried peptides were reconstituted in 12 μl of HPLC loading buffer (2% acetonitrile and 0.05% TFA in water) and approximately 1 μg peptides were analyzed by nanoLC-MS/MS using a Q-Exactive HF and Ultimate 3000 nanoHPLC system (Thermo Fisher Scientific, Waltham, MA, United States). Peptides were loaded onto a 20 mm PepMap 100 C18 trapping column (3 mm C18) at 5 μl min⁻¹. Using a 2 h segmented gradient from mobile phase A (0.1% formic acid in water) to mobile phase B [0.08% formic acid in acetonitrile/water (80:20)], peptides were separated at 300 nl min⁻¹ on a 250 mm PepMap 100 C18 analytical column at 45°C. For phosphopeptide analysis, the dried samples were reconstituted in 10 μl of HPLC loading buffer, of which 4  μl was injected then separated under the same conditions, using a shorter (1 h) segmented gradient. The data-independent acquisition mass spectrometry (DIA-MS) method used for both total peptide and phosphopeptide analysis has been previously described in Balotf et al. (2021). Raw MS files were processed using Spectronaut software (v15) using the directDIA approach. Spectral libraries were generated independently from the total peptide and phosphopeptide samples using the Pulsar search engine to search the Solanum tuberosum UniProt reference proteome (UP000011115) comprising 53,106 entries. Default search settings were used, with the exception that for phosphosite identification the PTM localization filter was activated (minimum threshold 0.75) and phospho (S/T/Y) was included as an additional variable modification. Relative quantitation between samples at the proteome or phosphoproteome levels was then achieved by targeted re-extraction of DIA-MS2 spectra from respective total peptide or phosphopeptide libraries. Data were exported from Spectronaut as a PTM site report for the phosphopeptide data and a protein group pivot report for the proteomics data set.