Identified unique proteins and peptides from HeLa Tryptic Digest injections on an Aurora FrontierTM 60 cm x 75 μm column using different gradient lengths and instruments.

Samples were either run on a Vanquish Neo UHPLC coupled to a Thermo Orbitrap Exploris 480 running a 90 min (1.5 μg injection; n = 5) or 180 min (3 μg injection; n = 4) sample gradient (300 nL/min), or a U3000 UHPLC coupled to a Thermo Orbitrap Exploris 240 +FAIMS Duo running a 180 min (3 μg injection; n = 4) sample gradient (200 nL/min).

Samples were acquired using a DIA workflow and searched using DIA-NN (1% FDR) using an 8 fraction library created using small m/z window DIA acquisitions between m/z 380 and 985. Unique proteins counted at the Protein level.