Multiple and Sequential
Data Acquisition Method: An
Improved Method for Fragmentation and Detection of Cross-Linked Peptides
on a Hybrid Linear Trap Quadrupole Orbitrap Velos Mass Spectrometer
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Abstract
The identification and validation of cross-linked peptides
by mass
spectrometry remains a daunting challenge for protein–protein
cross-linking approaches when investigating protein interactions.
This includes the fragmentation of cross-linked peptides in the mass
spectrometer per se and following database searching, the matching
of the molecular masses of the fragment ions to the correct cross-linked
peptides. The hybrid linear trap quadrupole (LTQ) Orbitrap Velos combines
the speed of the tandem mass spectrometry (MS/MS) duty circle with
high mass accuracy, and these features were utilized in the current
study to substantially improve the confidence in the identification
of cross-linked peptides. An MS/MS method termed multiple and sequential
data acquisition method (MSDAM) was developed. Preliminary optimization
of the MS/MS settings was performed with a synthetic peptide (TP1)
cross-linked with bis[sulfosuccinimidyl] suberate (BS<sup>3</sup>).
On the basis of these results, MSDAM was created and assessed on the
BS<sup>3</sup>-cross-linked bovine serum albumin (BSA) homodimer.
MSDAM applies a series of multiple sequential fragmentation events
with a range of different normalized collision energies (NCE) to the
same precursor ion. The combination of a series of NCE enabled a considerable
improvement in the quality of the fragmentation spectra for cross-linked
peptides, and ultimately aided in the identification of the sequences
of the cross-linked peptides. Concurrently, MSDAM provides confirmatory
evidence from the formation of reporter ions fragments, which reduces
the false positive rate of incorrectly assigned cross-linked peptides