Workflow of the study. (A) MPM candidate biomarker glycopeptides were identified from the surfaceome of model cell lines (1). Detection in serum was targeted verified by SRM assay technology (2). Diagnostic significance was investigated by multiplexed SRM assay technology in clinical cohorts (3). (B) CSC technology was applied for the quantitative surfaceome screening of four MPM, two non-cancerous pleural and two NSCLC (lung adenocarcinoma) cell lines for MPM candidate biomarker N-glycopeptides (Additional file 4: Table S3) (1). SRM-assays were generated for the MPM candidate biomarkers and applied for the screening of five MPM sera (Additional file 7: Table S4 and Additional file 5: Skyline file) (2). SRM-assays for MPM candidate biomarkers detected in serum were multiplexed in one single SRM-method and quantitatively evaluated in a clinical training set of enriched sera from 25 MPM, 25 HD and 25 NSCLC (Additional file 8: Table S5 and Additional file 9: Table S6) (3). Candidate biomarkers detected at higher abundance in sera of MPM were combined in logistic regression models to derive a multiplexed panel of six glycopeptides with optimal accuracy in discriminating MPM from HD. The panel was further confirmed in an independent validation set of 30 MPM, 29 HD and 28 NSCLC (Additional file 8: Table S5) together with the SRM-based monitoring of the biomarker mesothelin (Additional file 11: Table S7).