xComb: a cross-linked peptide database approach to protein-protein interaction analysis

Abstract

We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral data sets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community.

Figure 1. Description of the xComb strategy

A.1) All theoretically possible cross-linked amino acid combinations are built based on the input protein sequences. Irrelevant combinations are then filtered based on specific parameters defined by the user (e.g. cross-linker specificity). A.2) A cross-linked peptides fasta database is generated with each entry corresponding to one cross-linking combinations (both permutations are written). A header describes precisely both peptides (origin and position) for straightforward interpretation and validation. B) Product ion spectra are acquired on ≥+4 precursors only at high mass accuracy. Depending on the search engine used, raw tandem mass spectral data are first de-convoluted and re-written into a lower charge state form. C) Finally, tandem mass spectra can be searched against the cross-linked peptides database using any standard search engine (Sequest, Phenyx, Mascot, X!Tandem or OMSSA). A “do not cleave” enzyme is added and used to specifically search only the full length cross-linked sequences. The cross-link reagent is specified as a variable modification with the addition of a water molecule to account for loss of mass during the linearization process.

Figure 2. Description of the linearization process

Two peptides cross-linked together can be considered as two unique linearized forms of both sequences that cover 100% of the fragment ions. These two concatenated sequences may be directly interpreted by a standard search engine to cover all fragment ions present in the spectrum.

Figure 3. Example of an identification based on only one permutation in the cytochrome P450 2E1/cytochrome b5 complex

A) Aspartic acid D₆ of peptide α is cross-linked to lysine K₄ of peptide β. The linearization αβ depicted here covers 80% of the fragment ions. B) and C) Spectrum matching to the linearization αβ. Almost all fragment ions are assigned to this spectrum with a high z-Score using Phenyx. The extent of fragment assignment allows validation of the position of the cross-linked amino acid. Unlike linearization αβ, βα poorly matches to the spectrum with a low z-Score of 3.56 (data-not shown).