Proteomics Analysis of Islet Samples

A Dionex UltiMate 1000 system (Thermo Scientific) was coupled to an Orbitrap Fusion Lumos (Thermo Scientific) through an Easy-Spray ion source (Thermo Scientific). For the islet sample, nLC separation was carried out as follows, while other samples were analyzed as previously described^{21 (link)}. Peptide samples were loaded (15 μL/min, 1 min) onto a trap column (100 μm × 2 cm, 5 μm Acclaim PepMap 100 C18, 50 °C), eluted (0.2 μL/min) onto an Easy-Spray PepMap RSLC C18 column (2 μm, 50 cm × 75 μm ID, 50 °C, Thermo Scientific) and separated with the following gradient, all % Buffer B (0.1% formic acid in ACN): 0–110 min, 2%–22%; 110–120 min, 22%–35%; 120–130 min, 35–95%; 130–150 min, isocratic at 95%; 151–153 min, 95%–2%, 153–171 min, isocratic at 2%. Spray voltage was 1900V, ion transfer tube temperature was 275°C, and RF lens was 30%. MS scans were acquired in profile mode and MS/MS scans in centroid mode, for ions with charge states 2–7, with a cycle time of 3 sec. MS spectra were recorded from 375–1500 Da at 120K resolution (at m/z 200), and HCD MS/MS was triggered above a threshold of 2.0e4, with quadrupole isolation (0.7 Da) at 30K resolution, and collision energy (CE) of 30%. Dynamic exclusion was used (60 s), and monoisotopic precursor selection was on.