Updated FTP location. Single-shot proteome analysis of HeLa cell lysate with 6-h gradient using a 1-m spray needle column (Waters ACQUITY UPLC BEH C18, 1.7 µm, ID = 75 µm, Tip ID = 3 µm) and a nanoUPLC-MS system (Bruker nanoElute/timsTOF Pro). The column was prepared in-house by our developed approach (syringe suction method), and the analytical performance in deep shotgun proteomics was demonstrated in this project.