We propose an efficient integration of SWATH with MRM for biomarker discovery and verification when the corresponding ion library is well established. We strictly controlled the false positive rate associated with SWATH MS signals and carefully selected the target peptides coupled with SWATH and MRM. We collected 10 samples of esophageal squamous cell carcinoma (ESCC) tissues paired with tumors and adjacent regions and quantified 1758 unique proteins with FDR 1% at protein level using SWATH, in which 467 proteins were abundance-dependent with ESCC. After carefully evaluating the SWATH MS signals of the up-regulated proteins, we selected 120 proteins for MRM verification. MRM analysis of the pooled and individual esophageal tissues resulted in 116 proteins that exhibited similar abundance response modes to ESCC that were acquired with SWATH. Because the ESCC-related proteins consisted of a high percentile of secreted proteins, we conducted the MRM assay on patient sera that were collected from pre- and postoperation. Of the 116 target proteins, 42 were identified in the ESCC sera, including 11 with lowered abundances postoperation. Coupling SWATH and MRM is thus feasible and efficient for the discovery and verification of cancer-related protein biomarkers.

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