Kojak: efficient analysis of chemically cross-linked protein complexes

Abstract

Protein chemical cross-linking and mass spectrometry enable the analysis of protein-protein interactions and protein topologies; however, complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein-protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms and can identify cross-linked peptides using many different chemical cross-linkers with or without heavy isotope labels. Kojak was used to analyze both novel and existing data sets and was compared to existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms and, equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing data sets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods.

Keywords: cross-linking; mass spectrometry; protein structure; proteomics.

Figure 1

Flow diagram of the Kojak algorithm.

Figure 2

Example analysis of a cross-linked peptide spectrum using Kojak. (a) The MS/MS spectrum of a triply-charged precursor ion of m/z 751.07. The spectrum is processed (inset) to collapse isotope envelope distributions and facilitate fast and accurate peptide identification. (b) Matches to product ions for individually searched peptides are scored and ranked. p_n is the theoretical peptide mass and x_n is the modification mass of the internal lysine, which sum to the precursor mass. The top 250 (user-defined) scoring peptide sequences are then pairwise compared. Matches are made when two peptides plus the cross-linker mass sum to the precursor ion mass. (c) The best matching pair of peptides was the first and second ranked peptides from panel (b).

Figure 3

Comparison of the uniquely cross-linked sites for the CRY2-FBXL3-SKP1 complex.

Figure 4

Structure of the CRY2-FBXL3-SKP1 complex. CRY2 is shown in red, FBXL3 is shown in purple, and SKP1 is shown in green. (a) Surface representation of the overall complex structure (PDB code: 4I6J). (b) Ribbon diagram of the structure with cross-linked lysine residues superimposed. Cross-links were identified using Kojak with a 1% FDR cutoff. Yellow lines indicate Cα-Cα within 30 Å, and blue lines indicate Cα-Cα distances exceeding 30 Å.