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The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification,resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer (MS) running time.Here we report a fast sequencing (Fast-seq) workflow of the use of dual reversed phase HPLC-MS/MS with a short gradient to achieve the same proteome coverage in 0.5 day.