4.7. Mass Spectrometry

Fractions of dried peptides were re-dissolved in 35 µL 0.1% trifluoroacetic acid and analyzed individually. For this, peptides were loaded on a 75 µm × 2 cm Acclaim C18 precolumn (Thermo Fisher) using an RSLCnano HPLC system (Thermo Fisher). Then, peptides were eluted with an aqueous-organic gradient (4–44% acetonitrile, 0.1% formic acid) for 130 min and separated on a 75 µm × 15 cm Acclaim C18 column (Thermo Fisher) with a flow rate of 300 nl/min. A Triversa Automate (Advion, Ithaca, NY, USA) was used as the ion source to produce a stable electrospray, which was analyzed on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher). Each scan cycle consisted of 1 FTMS full scan and up to 10 ITMS dependent MS/MS scans of the 10 most intense ions with the dynamic exclusion set to 30 sec. The mass width was set to 10 ppm and monoisotopic precursor selection was enabled. All analyses were performed in positive ion mode.