Peptide Analysis by Nano-LC-MS/MS

Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the EASY-nLC 1000 system (Proxeon Biosystems, Odense, Denmark, now part of Thermo Fisher Scientific) with 20 cm capillary columns of an internal diameter of 75 μm filled with 1.8 μm Reprosil-Pur C18-AQ resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted with a linear gradient from 5-30% buffer B (80% acetonitrile in 0.1% formic acid) for 100 min, 30-60% B for 12 min and 80-95% B for 8 min at a flow rate of 250 nl/min. The eluate was electro-sprayed into an Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) using a Proxeon nanoelectrospray ion source. The Orbitrap Elite was operated essentially as previously described [62 (link)] in a HCD top 10 mode with dynamic selection of the 10 most intense peaks of each survey scan (300-1750Th) for fragmentation. The resolution was 120,000 for full scans and 15,000 for fragments (both specified at m/z 400). Ion target values were 1e6 and 5e4ms, respectively. Dynamic exclusion time was 30 sec.