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.