Easy column c18 trapping column

Digested proteins were analysed in technical triplicates by LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. Peptides were loaded on the EASY-Column C18 trapping column (2 cm L., 100 µm I.D, 5 µm ps, Thermo Fisher Scientific), and subsequently separated on an Acclaim PepMap100 C18 (75 µm I.D., 25 cm L, 5 µm ps, Thermo Fisher Scientific) nano scale chromatographic column. The flow rate was set to 300 nL/min and the gradient was from 3 to 35% of B in 80 min followed by 35 to 45% in 10 min and from 45 to 90% in 11 min. Mobile phase A was 0.1% formic acid in H₂O and mobile phase B was 0.1% formic acid in acetonitrile. The mass spectrometer was equipped with a nanoESI spray source and operated in positive ion polarity and Auto MS/MS mode (Data Dependent Acquisition - DDA). Precursors in the range 350 to 2,200 m/z (excluding 1,220.0–1,224.5 m/z) with a preferred charge state +2 to +5 (excluding singly charged ions). After acquiring one MS/MS spectrum, the precursors were actively excluded from selection for 30 seconds. In-source reference lock mass (1,221.9906 m/z) was acquired online throughout the runs.

A typical protocol of filter-aided sample preparation (FASP) for tryptic digestion was carried out overnight at 37 °C using trypsin (Promega, Madison, WI, USA) both for whole tears and FACS-sorted EVs. Digested proteins from each sample were analyzed in triplicate by LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. Digested peptides were analyzed on the EASY-Column C18 trapping column (2 cm L., 100 µm I.D, 5 µm ps, Thermo Fisher Scientific, Milan, Italy), and subsequently on an Acclaim PepMap100 C18 (75 µm I.D., 25 cm L, 5 µm ps, Thermo Fisher Scientific, Milan, Italy) The flow rate was set to 0.300 µL/min with a total run time of 90 min. Chromatographic parameters and mass spectrometer setting conditions have been reported in our previous work [14 (link)].