Proteomic Analysis of Frozen Peptides

Frozen peptides were reconstituted in 3% ACN and 0.1% formic acid. A pooled sample was created by mixing 1 μL of each sample for every experiment. All samples in the same experiment were subjected together in a random order to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) for proteomic analysis as described previously⁶² (link) with the modification described below. In brief, peptides were first separated using a Thermo Scientific EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to a 15 cm-long and 75 μm-diameter silica emitter as well as a ReproSil-Pur C18-AQ 120 A and 1.9 μm resin (Dr Maisch HPLC GmbH). A three-liner gradient of acetonitrile/water (containing 0.1% formic acid, at a flow rate of 300 nL/minute), first from 2% to 30% acetonitrile in 60 min, second from 30% to 97% in 10 minutes, then 97% for 10 min, was applied. The mass spectrometer was set in a data-dependent manner with an automatic switch between MS and MS/MS using the Xcalibur software package (Thermo Fisher Scientific). A mass range of 300–1500 m/z was selected for the Orbitrap analyzer.