147 A novel liquid nitrogen-free snap freezer g, 4 min). Phosphopeptides were desalted using a 200 µl STAGE tip containing a 16G empore SDB-XC membrane plug (3 M, St Paul, MN) using the same solvents as used for the Seppak cartridge (20 µl, 100 rpm, 1 min). Desalted phosphopeptides were dried in a vacuum centrifuge and redissolved in 20 µl 4% ACN, 0.5% TFA; 17 µl was injected on the column. Phosphopeptides were separated by nanoLC and detected as described elsewhere 2,4,5 on a Q exactive HF mass spectrometer (Thermo Fisher, Bremen, Germany). PROTEIN IDENTIFICATION LC-MS/MS spectra were searched against the Uniprot human reference proteome FASTA file (release February 2019, 42417 entries, no fragments) using MaxQuant 1.6.4.06. Enzyme specificity was set to trypsin and up to two missed cleavages were allowed. Cysteine carboxamidomethylation (Cys, +57.021464 Da) was treated as fixed modification and serine, threonine and tyrosine phosphorylation (+79.966330 Da), methionine oxidation (Met, +15.994915 Da) and N-terminal acetylation (N-terminal, +42.010565 Da) as variable modifications. Peptide precursor ions were searched with a maximum mass deviation of 4.5 ppm and fragment ions with a maximum mass deviation of 20 ppm. Peptide, protein and site identifications were filtered at a false discovery rate (FDR) of 1% using the decoy database strategy. The minimal peptide length was 7 amino acids and the minimum Andromeda score for modified peptides was 40, with the corresponding minimum delta score set at 177. Proteins that could not be differentiated based on MS/MS spectra alone were grouped into protein groups (default MaxQuant settings). (Phospho)peptide identifications were propagated across samples using the match-between-runs option checked. Searches were performed with the label-free quantification option selected. (Phospho)peptide identifications were propagated across samples using the match-between-runs option checked. Searches were performed with the label-free quantification option selected. LABEL-FREE PHOSPHOPEPTIDE QUANTIFICATION Phosphopeptides were quantified by their extracted ion chromatograms (‘Intensity’ in MaxQuant). For each sample the phosphopeptide intensities were normalized on the median intensity of all identified peptides in the sample (‘normalized intensity’ from the MaxQuant Evidence table). Normalization and statistical testing were performed in R. Fold-change and p values were calculated from replicates using a two-tailed Student’s t-test; phosphopeptides were considered significantly differential at p < 0.05. The match-between-runs option in MaxQuant was used. Missing values were excluded from subsequent statistical analysis. Quantitative values from replicates were averaged prior to biological group comparisons. The t-test requires at least two quantitative values in each group. P-values were not corrected for multiple hypothesis testing. Cluster analysis of differential phosphopeptides was performed using hierarchical clustering in R and repeated for the top10 and 20% most variable peptides. Phosphopeptide intensities were normalized to zero mean and unit variance for each phosphopeptide. Subsequently, the Euclidean distance measure was used for phosphopeptide clustering. For sample clustering metrics, the (1-Pearson correlation) distance and the Ward linkage were used. 5
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