Improved Analysis of Cross-Linking Mass Spectrometry Data with Kojak 2.0, Advanced by Integration into the Trans-Proteomic Pipeline

Fragmentation ion spectral analysis of chemically cross-linked
proteins is an established technology in the proteomics research repertoire
for determining protein interactions, spatial orientation, and structure.
Here we present Kojak version 2.0, a major update to the original
Kojak algorithm, which was developed to identify cross-linked peptides
from fragment ion spectra using a database search approach. A substantially
improved algorithm with updated scoring metrics, support for cleavable
cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0
presented here. Kojak 2.0 is now integrated into the Trans-Proteomic
Pipeline, enabling access to dozens of additional tools within that
suite. In particular, the PeptideProphet and iProphet tools for validation
of cross-links improve the sensitivity and accuracy of correct cross-link
identifications at user-defined thresholds. These new features improve
the versatility of the algorithm, enabling its use in a wider range
of experimental designs and analysis pipelines. Kojak 2.0 remains
open-source and multiplatform.