Phosphoproteomic interrogation identifies novel activated protein pathways in primary murine HSPCs. Resting BM (rest) or mobilized (mob) LSK HSPCs were harvested and sorted as described in “Methods.” (A) Comprehensive phosphoproteomic analysis of 2 × 105 primary murine resting or mobilized HSPCs was done in triplicate, and this experiment was repeated 3 times. In total, this analysis identified 15 230 unique phosphopeptides and 4993 phosphoproteins. The phosphopeptide ratios followed a normal distribution and there was not a marked change in total phosphopeptide quantity with mobilization. Overall, 1018 phosphopeptides differed in relative amount by >2 SD between mobilized and resting cells. Of these, 572 phosphopeptide species were >2 SD more abundant in mobilized HSPCs (red), and 446 were >2 SD less abundant (green). Red trace shows Gaussian overlay. (B) Unsupervised hierarchical clustering analysis of all 18 samples demonstrates that mobilization results in durable phosphoproteomic changes in primary murine HSPCs. Mob1/rest1, mob2/rest2, and mob3/rest3 represent individual experiments; a, b, and c denote biological replicates within experiments. Sample phosphoprofiles consistently clustered with other samples in their biologic subgroup (mobilized or resting) and away from samples in the other subgroup, confirming the existence of a durable phosphoproteomic signature of mobilization. (C) NMF of data from all 3 experiments identifies two phosphoproteomic signatures that can be used to segregate mobilized from resting HSPCs. Using the nonsmooth NMF method of Pascual-Montano et al, 1250 iterations were run with a factorization rank of 2 to identify consensus clusters capable of segregating mobilized from resting phosphoprofiles.26 Heatmap shows the degree of concordance between sample groups (top bars) and consensus profiles (left bars). (D) Signature phosphoprotein residues were extracted and then filtered by featurescore to identify phosphoprotein residues most specific to each signature. A mixture expression profile heatmap, which summarizes the relative contribution of each signature to each sample was used to assign signatures, or metagroups (left), to the sample classes (top). A complete list of phosphoprotein residues in each metagroup is provided in supplemental Table 3.
