A study aimed at identification of proteins associated with CRISPR/Cas-induced depletion of creatine kinase B (CKB) in SAOS-LM5 metastatic osteosarcoma cell line using LC-MS/MS in data-independent acquisition (DIA)-parallel accumulation serial fragmentation (diaPASEF) mode. Here we prepared two KO cell lines, SAOS-LM5 CKB KO 6C and SAOS-LM5 CKB KO 11B, as well as control SAOS-LM5 scrambled cell line. After filter-aided sample preparation (FASP) protein digestion and peptide desalting, the samples were conducted to total proteome analysis. The data acquisition was performed in diaPASEF mode on timsTOF Pro 2 mass spectrometer. Data were processed in Spectronaut 19.0 in directDIA mode. This project covers the raw files from the diaPASEF measurements as well as search results from the Spectronaut 19.0 software.