LC-MS/MS Analysis of Peptide Extracts

LC-MS/MS analysis was performed as described earlier [77 (link)]. Dried peptide extracts were dissolved in 100 µL of 0.1% formic acid and analyzed on a nano-HPLC system (LC-20nano, Shimadzu; Vienna, Austria). Then, 50 µL samples were injected and concentrated on the loading column (LC Packings C18 Pep- Map™, 5 µm, 100 Å, 300 µm inner diameter × 1 mm) for 5 min using 0.1% formic acid as isocratic solvent at a flow rate of 20 µL/min. The column was then switched into the nanoflow circuit, and the sample was loaded on the nanocolumn (LC-Packings C18 PepMap™, 75 µm inner diameter × 150 mm) at a flow rate of 300 nL/min and separated using the following gradient: solvent A: water, 0.3% formic acid, solvent B: acetonitrile/water (80/20, v/v), 0.3% formic acid; 0 to 5 min: 4% B, after 40 min 55% B, then for 5 min 90% B and 47 min reequilibration at 4% B. The sample was ionized in a Finnigan nano-ESI source equipped with NanoSpray tips (PicoTip™ Emitter, New Objective, Woburn, MA, USA) and analyzed in a Thermo-Finnigan LTQ linear iontrap mass-spectrometer (Thermo, San Jose, CA, USA). MS/MS data were analyzed by searching the SwissProt public database with SpectrumMill Rev. B.04.01.141 (Agilent, Darmstadt, Germany) software. Acceptance was a protein score of >20 and individual peptide scores >7.5.