Data-independent acquisition (DIA) mass spectrometry was used to study the effect of JAG1 protein in ECM organization in breast cancer. TNBC cell line samples including MDA-MB-231 cells (Jag1WT), JAG1 knock-out of MDA-MB-231 cells (Jag1KO), empty vector transfected to Jag1KO, and JAG1 rescued to Jag1KO cocultured with mouse embryonic fibroblasts (MEFs) as spheroids were used to compare the ECM proteins by mass spectrometry.