Tonic signaling of the B‐cell antigen‐specific receptor is a common functional hallmark in chronic lymphocytic leukemia cell phosphoproteomes at early disease stages

B‐cell chronic lymphocytic leukemia (B‐CLL) is characterized by highly heterogeneous genomic alterations and altered signaling pathways, with limited studies on its proteome. Our study presents a comprehensive analysis of the proteome and phosphoproteome in B‐CLL and CLL‐like monoclonal B‐cell lymphocytosis (MBL) primary cells. Using high‐resolution mass spectrometry, we identified 2970 proteins and 316 phosphoproteins across five tumor samples, including 55 newly identified phosphopeptides (ProteomeXchange‐PXD005997). Our multifaceted approach also integrated protein microarrays and western blotting for further data validation in a new patient cohort of 14 patients. Despite sharing 73% of their proteomes, the phosphoproteomes varied significantly among samples, independent of cytogenetic alterations and immunoglobulin heavy variable cluster (IGHV) mutational status. We identified common functional hallmarks in B‐CLL and MBL phosphoproteomes, notably tonic signaling (low‐level, constitutive signaling) of the B‐cell antigen‐specific receptor (BCR) and nuclear factor NF‐kappa‐B (NF‐kβ)/signal transducer and activator of transcription 3 (STAT3) pathways. Nine phosphoproteins involved in BCR signaling were further validated, showing a high correlation with early disease stages. Our study advances the field by providing a detailed perspective on the proteome and phosphoproteome of B‐CLL cells, revealing signaling pathways crucial for disease development and progression. Integrating diverse proteomics techniques and identifying novel phosphopeptides offers new insights into CLL biology, potentially informing future therapeutic strategies and biomarker development for early diagnosis and personalized treatment.