Optimizing the Parameters Governing the Fragmentation of Cross-Linked Peptides in a Tribrid Mass Spectrometer

We compared the five
different ways of fragmentation available
on a tribrid mass spectrometer and optimized their collision energies
with regard to optimal sequence coverage of cross-linked peptides.
We created a library of bis­(sulfosuccinimidyl)­suberate (BS3/DSS) cross-linked
precursors, derived from the tryptic digests of three model proteins
(Human Serum Albumin, creatine kinase, and myoglobin). This enabled
in-depth targeted analysis of the fragmentation behavior of 1065 cross-linked
precursors using the five fragmentation techniques: collision-induced
dissociation (CID), beam-type CID (HCD), electron-transfer dissociation
(ETD), and the combinations ETciD and EThcD. EThcD gave the best sequence
coverage for cross-linked m/z species
with high charge density, while HCD was optimal for all others. We
tested the resulting data-dependent decision tree against collision
energy-optimized single methods on two samples of differing complexity
(a mix of eight proteins and a highly complex ribosomal cellular fraction).
For the high complexity sample the decision tree gave the highest
number of identified cross-linked peptide pairs passing a 5% false
discovery rate (on average ∼21% more than the second best,
HCD). For the medium complexity sample, the higher speed of HCD proved
decisive. Currently, acquisition speed plays an important role in
allowing the detection of cross-linked peptides against the background
of linear peptides. Enrichment of cross-linked peptides will reduce
this role and favor methods that provide spectra of higher quality.
Data are available via ProteomeXchange with identifier PXD006131.