IntroductionGastric cancer is a highly heterogeneous malignant tumor of the digestive system. Anti-HER2 treatment can inhibit downstream signaling pathways and improve clinical treatment and outcomes in patients with HER2 protein overexpression. Currently, two standard methods for evaluating HER2 expression status are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, these low-throughput assays often produce discordant or equivocal results.MethodsIn this study, we presented a new HER2 protein detection method based on mass spectrometry selected reaction monitoring (MS-SRM) and validated the method. We conducted a retrospective study on 118 formalin-fixed paraffin-embedded (FFPE) tissues from patients with advanced gastric adenocarcinoma in northern China, and we compared the MS-SRM results with those from IHC and correlated them with FISH.ResultsWe established and validated the upper and lower detection limits (300-700 amol/μg) for abnormal HER2 protein expression in advanced gastric cancer. We also found that, among samples with mixed Lauren subtypes, those with a high level of HER2 expression had typical intestinal type features in pathology.DiscussionThis study demonstrated that the MS-SRM method can overcome the limitations and deficiencies of IHC, directly quantify the expression of HER2 protein in tumor cells and be used as a supplement to IHC. It has the potential to be used as a companion diagnosis for new drugs used to treat advanced gastric cancer. Large-scale clinical validation is required.