Only peptides identified with full confidence were included in the analysis (binomial probability threshold of P < 10−6, occurrence threshold = 20). Each biological replicate was median-adjusted to 10 to account for differences in sample loading, resulting in the following changes: proteomic data set: P1A, −0.0362; P1B, −0.0879; P1C, +0.20476; P7A, +0.03374; P7B, −0.2761; P7C, −0.2865; P14A, +0.78605; P14B, +0.65244; P14C, +0.71771; MB, +0.04692; phosphoproteomic data set: P1A, −0.040163003; P1B, −0.124877212; P1C, −0.009654128; P7A, −0.170361415; P7B, −0.341821765; P7C, −0.117111143; P14A, +1.114888992; P14B, +1.060676918; P14C, +0.955615374; MB, +0.43299154. Log2-transformed P1:P7 and P7:P14 ratios were determined, and phosphopeptide changes were normalized to protein changes. Values outside of 1.5 SD were considered “significant changers.” Motif analysis of significantly changing phosphopeptides was performed using Motif-X, as previously described (61, 62). Briefly, Motif-X is an iterative statistical approach to identifying protein phosphorylation motifs in large-scale phosphoproteomic data sets built on a greedy recursive search of the sequence space to identify highly correlated residue/position pairs with the lowest P values. Here, a binomial probability threshold of P < 10−6 and occurrence threshold of 20 were used.

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