Thermo ltq velos

Dry peptide fractions were resuspended in 0.1% FA and loaded onto a Thermo Easy-LC system. Peptides were eluted using a 0–34% organic gradient over 70 min, then 34–100% over 20 min. All LC-MS runs were performed on columns with a 75-μm inner diameter, packed with C18 material (Dr. Maisch, Ammerbuch-Entringen, Germany). Mass spectrometry (MS) was performed using higher-energy collisional dissociation (HCD) fragmentation on a Thermo LTQ Velos (Thermo Fisher Scientific, Germany). Quantification was performed on reporter tags at m/z 114, 115, 116, and 117. MS was performed with the following settings: A full MS scan in the mass area of 400–1800 Da was performed in the Orbitrap with a resolution of 30,000 FWHM and a target value of 1 × 10⁶ ions. For each full scan, the seven most intense ions (>+1 charge state) were selected for HCD and detected at a resolution of 7500 FWHM. HCD was performed with the following settings: threshold for ion selection was 20,000, the target value of ions used for HCD was 1 × 10⁵, and activation time was 1 ms, the isolation window was 2.5 Da, and the normalized collision energy was 48.

The samples were analyzed by reversed phase nanoflow liquid chromatography and tandem mass spectrometry (LC-MS/MS) using an Easy nLC-II system (Thermo, San Jose, CA) coupled to a Thermo LTQ Velos dual pressure linear ion trap system (Thermo, San Jose, CA). Standard equine cytochrome C digest was injected as a quality control. Two microliters sample was loaded via the autosampler onto a trap column (EASY-Column 2 cm, ID 100 µm, 5 µm, C18-A) then directed to an analytical column (EASY-Column, 10 cm, ID 75 µm, 3 µm, C18-A2) at a flow rate of 250 nl/min. The mobile phase consisted of solvent A (99.9% water with 0.1% formic acid) and solvent B (99.9% acetonitrile with 0.1% formic acid). Separation was achieved using a run time of 100 min. The first linear gradient was from 2% to 40% B over 90 min, the second linear gradient was from 40% to 80% B over 5 min and held for 5 min before returning to the initial mobile phase composition (2% B). Tandem mass spectra (MS/MS) were acquired on the top 10 most abundant ions at a given chromatographic time point by data-dependent scanning.