Ultraviolet Photodissociation of Tryptic Peptide Backbones at 213 nm

We
analyzed the backbone fragmentation behavior of tryptic peptides
of a four-protein mixture and of E. coli lysate subjected
to ultraviolet photodissociation (UVPD) at 213 nm on a commercially
available UVPD-equipped tribrid mass spectrometer. We obtained 15 178
unique high-confidence peptide UVPD spectrum matches by recording
a reference beam-type collision-induced dissociation (HCD) spectrum
of each precursor, ensuring that our investigation includes a broad
selection of peptides, including those that fragmented poorly by UVPD.
Type a, b, and y ions were most prominent in UVPD spectra, and median
sequence coverage ranged from 5.8% (at 5 ms laser excitation time)
to 45.0% (at 100 ms). Overall, the sequence fragment
intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100
ms) of total intensity), and the remaining precursor intensity, high.
The sequence coverage and sequence fragment intensity ratio correlated
with the precursor charge density, suggesting that UVPD at 213 nm
may suffer from newly formed fragments sticking together due to noncovalent
interactions. The UVPD fragmentation efficiency therefore might benefit
from supplemental activation, as was shown for ETD. Aromatic amino
acids, most prominently tryptophan, facilitated UVPD. This points
to aromatic tags as possible enhancers of UVPD. Data are available
via ProteomeXchange with identifier PXD018176 and on spectrumviewer.org/db/UVPD-213nm-trypPep.