Chromatographic separation was performed on a Bruker PepSep column (0.15x150 mm, ReproSil-Pur C18-AQ 1.9 μm, 120 Å, C-load 15%; Billerica, MA, USA) in a Waters M-Class system composed of a μBSM binary pump with an embedded flow controller, a μSM-FL fixed loop sampler and a TVM module equipped with switching valves and a heated column compartment. Injection volume was set to 0.2 μL using partial loop direct injection mode. The calculated delay volume from the flow controller outlet to the column inlet was 1.76 μL with all the fluidic connections created using SecurityLink fittings (25 μm, Phenomenex, Torrance, CA, USA). Mobile phases were (A) acetonitrile:water + 0.1% acetic acid (1:1 v/v, 10 mM ammonium acetate) and (B) isopropanol:acetonitrile + 0.1% acetic acid (8:2 v/v, 10 mM ammonium acetate). The gradient program ran at 3 μL/min from 3 to 99% B in 11.5 min for a total run time of 19 min. The detection was carried out using an online hyphenated Orbitrap Exploris 120 mass spectrometer (ThermoFisher Scientific) equipped with a DuoESI source containing a 5-nozzle (20 μm/nozzle) or an 8-nozzle (10 µm/nozzle) M3 silicon emitter (Newomics, Berkeley, CA, USA). Connection of the column outlet to the emitter was performed using a 35 cm nanoViper capillary (20 μm, ThermoFisher Scientific) according to the manufacturer's instructions. The analysis was performed in positive polarity (4.5 kV for the 5-nozzle emitter and 3.5 kV for the 8-nozzle emitter) in DDA mode over a 400-1200 Da scan range and resolution set to 60'000 for MS 1 and 15'000 for MS 2 at m/z 200. Sheath gas was set at 6 a.u., while auxiliary and sweep gases were turned off. The transfer tube temperature was set at 300 °C, the RF lens at 70%, the ITT at 80 ms and the AGC at 200%. Stage position was set at 0 mm on X-axis, +2.7 mm on Y-axis (5-nozzle emitter) or + 3.5 (8-nozzle emitter) and Z-axis slightly adjusted to put the emitter in the same plane as MS inlet. Targeted mass exclusion list for MS/MS acquisition was based on lists provided by Keller et al. and M. J. Rardin . Full description of the method parameters is provided in supplementals (Table for the µF/mnESI and S3 for the AF/ESI). The analytical sequence used for batch-proofing the μF/mnESI method was constituted of 42 biological samples, 8 pooled QCs and 8 twofold diluted QCs, with a system conditioning consisting of 20 injections of QC material. To investigate the influence of injecting diluted material on the following sample, each diluted QC was followed by a re-conditioning pooled QC (μF/mnESI and AF/ESI sequences are detailed in supplemental Table ).