Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer

Dilute the peptide sample with solvent A (2% ACN, 0.1% FA) to the desired concentration. Transfer the samples to an autosampler vial and add iRT peptides to the sample according to the manufacturer’s instructions.

Inject the sample onto a Dionex UltiMate 3000 liquid chromatographic system coupled to an Orbitrap Eclipse Tribrid mass spectrometer and load the peptides on a C₁₈ trap column over 10 min at 5 µL/min with 100% solvent A.

Elute the peptides with a C₁₈ analytical column heated at 35°C and at a flow rate of 300 nL/min over 215 min using the following gradient of solvent B:

2% for 10 min

From 2% to 20% in 125 min

From 20% to 32% in 40 min

From 32% to 80% in 1 min

80% for 9 min

From 80% to 2% in 1 min

2% for 29 min.

Acquire the data in data-independent acquisition (DIA) mode using the settings described in Table 1 . One cycle consists of one full MS scan collected in the Orbitrap analyzer followed by DIA-MS/MS scans collected in the Orbitrap analyzer based on an isolation scheme of 26 variable windows covering the 350 - 1,650 m/z range with an overlap of 1 m/z ( Table 2 ).