Eksigent nanolc ultra 1d

Nanoflow LC-MS/MS was performed by coupling an Eksigent nanoLC-Ultra 1D + (Eksigent, Dublin, CA) to a LTQ-Orbitrap XL ETD (Thermo Scientific, Bremen, Germany). Peptides were delivered to a trap column (100 μm × 2 cm, packed in-house with Reprosil-Pur C₁₈-AQ 5 μm resin, Dr. Maisch, Ammerbuch, Germany) at a flow rate of 5 μL/min in 100% solvent A (0.1% formic acid in HPLC grade water). After 10 min of loading and washing, peptides were transferred to an analytical column (75 μm × 40 cm, packed in-house with Reprosil-Pur C₁₈-GOLD, 3 μm resin, Dr. Maisch, Ammerbuch, Germany) and separated using a 210 min gradient from 4% to 32% of solvent B (0.1% formic acid, 5% DMSO in acetonitrile; solvent A: 0.1% formic acid, 5% DMSO in water) at 300 nL/minute flow rate. The LTQ Orbitrap XL was operated in data dependent mode, automatically switching between MS and MS [2 (link)]. Full scan MS spectra were acquired in the Orbitrap at 60,000 (m/z 400) resolution after accumulation to a target value of 1,000,000. Tandem mass spectra were generated for up to eight peptide precursors in the linear ion trap by using collision-induced dissociation at a normalized collision energy of 35% after accumulation to a target value of 5,000 for max 100 ms.

Kinobeads eluates were measured using nanoflow LC‐MS/MS by directly coupling an Eksigent nanoLC‐Ultra 1D+ (Eksigent) to an Orbitrap Elite mass spectrometer (Thermo Fisher). Chromatography as well as data acquisition was similar to the settings used for full proteome fractions; hence, we only describe differences between the two set‐ups. We injected 10 μl per measurement instead of 5 μl and used a slightly steeper gradient from 2% buffer B to 4% in 2 min and from 4 to 32% in 88 min instead of 96 min. The rest of the gradient was kept the same, resulting in a turnaround time of 110 min per Kinobeads pulldown, totalling ~15 days of measurement time for the entire CRC65 cell line panel in biological triplicates.