Analysis of proteomes and p-proteomes in M-CLL and UM-CLL cells. (A) Experimental setup for the mass spectrometric analysis of proteins and phosphoproteins from primary CLL samples involving iTRAQ labeling (proteome) and TiO₂ enrichment plus HILIC fractionation (p-proteome). Phosphoproteomic analysis was performed under 4 different conditions: (1) native, (2) stimulation with anti-IgM beads, (3) ibrutinib (Ibr) treatment, and (4) ibrutinib treatment plus stimulation with anti-IgM beads. Proteome analysis was performed in untreated cells. (B) Number and regulation of isolated proteins and phosphopeptides. (C) Number of proteins with distinct expression in untreated UM-CLL (vs M-CLL). (D) Volcano plot of protein expression in untreated UM-CLL vs M-CLL cells (gray, proteins associated with cell death and survival; green, proteins associated with cell migration and motility). Negative log₂ fold change: lower expression in UM-CLL, higher expression in M-CLL; positive log₂ fold change: higher expression in UM-CLL, lower expression in M-CLL. (E-F) Heat maps representing expression levels of the most relevant regulated proteins in untreated UM-CLL vs M-CLL. The ratio of UM-CLL to M-CLL was calculated using the 3 biological replicates per group.