LC-MS/MS with UVPD fragmentation
Peptide fragmentation by UVPD was carried out in an Orbitrap Eclipse Tribrid mass spectrometer (Thermofisher Scientific, Waltham, MA). Data were acquired by the DDA method and Orbitrap detection. MS1 was collected in the mass range from m/z 375 to m/z 1500 at a 120,000 resolution with an AGC target of 1 x 106 and a maximum injection time of 100 ms. Followed by MS1, MS2 was collected at a 30,000 resolution with an AGC target of 1 x 105 and a maximum injection time of 55ms. Each precursor was isolated with a 2.0 Th window. The peptides were fragmented by laser irradiation in the central part of the quadrupolar dual linear ion trap, and the fragment ions were transferred to the Orbitrap mass analyzer for measurement of their molecular masses. Peptides were photoactivated during 500 ms with the fifth harmonic Q-switched 213 nm Nd:YAG laser (CryLaS, GmbH). Peptides were applied to the Vanquish Neo UHPLC system (Thermo Fisher Scientific, Waltham, MA), and separated on a 75um ID x 125mm capillary column (Nikkyo Technos, Tokyo, Japan) with a linear gradient from 0% to 45% of solvent A/B (A : water with 0.1% formic acid; B : 80% acetonitrile with 0.1% formic acid (Thermo Fisher Scientific, Waltham, MA)) for 10 minutes (synthetic peptides) or 60 minutes (conjugates) at a flow rate of 300 nL/min and sprayed into the mass spectrometer with a 2000 V spray voltage as the ESI.