Mass spectrometry (MS)-based global proteomic data was used to developed a novel computational ranking to assess all cell adhesion molecule candidates in breast cancer. Cell lysate was processed using S-Trap workflow. MDA-MB-231 cell lysate were resuspended by 5% SDS. The protein concentration was measured with BCA protein assay and reduced with 10 mM DTT for 15 mins at 37 °C; subsequently alkylated with 50 mM iodoacetamide at 25 °C for 15 mins in the dark. The samples were acidified by phosphoric acid (the final concentration was 2.5%) and then diluted with six volumes of “binding” buffer (90% methanol; 100 mM triethylammonium bicarbonate, TEAB; pH 7.1). After mixing, the protein solution was loaded to an S-Trap filter from Protifi (Huntington, NY), spun at 10,000 g for 1 min and then the filter was washed with 150 μL of binding buffer 3 times. Proteins were digested with Lys-C (Wako) and sequencing-grade trypsin (Promega, V5117) in the filter at 37 °C for 16 h. To elute peptides, 40 μL of 50 mM TEAB, 40 μL of 0.2% formic acid in H2O, and 40 μL of 50% acetonitrile in H2O were added sequentially. The peptide solutions were pooled for BCA assay to estimate peptides amount. 20 μg of peptides were dried with SpeedVac and stored at −80 °C until LC-MS/MS analysis. Lyophilized peptides were reconstituted in 200 μL of 0.1% TFA with 2% ACN containing 0.01% DDM and analyzed by LC-MS/MS using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific) connected to a nanoACQUITY UPLC system (Waters) in DIA-MS/MS mode.