CrossSearch, a user-friendly search engine for detecting chemically cross-linked peptides in conjugated proteins

Abstract

Chemical cross-linking and high resolution MS have been integrated successfully to capture protein interactions and provide low resolution structural data for proteins that are refractive to analyses by NMR or crystallography. Despite the versatility of these combined techniques, the array of products that is generated from the cross-linking and proteolytic digestion of proteins is immense and generally requires the use of labeling strategies and/or data base search algorithms to distinguish actual cross-linked peptides from the many side products of cross-linking. Most strategies reported to date have focused on the analysis of small cross-linked protein complexes (<60 kDa) because the number of potential forms of covalently modified peptides increases dramatically with the number of peptides generated from the digestion of such complexes. We report herein the development of a user-friendly search engine, CrossSearch, that provides the foundation for an overarching strategy to detect cross-linked peptides from the digests of large (>or=170-kDa) cross-linked proteins, i.e. conjugates. Our strategy combines the use of a low excess of cross-linker, data base searching, and Fourier transform ion cyclotron resonance MS to experimentally minimize and theoretically cull the side products of cross-linking. Using this strategy, the (alpha beta gamma delta)(4) phosphorylase kinase model complex was cross-linked to form with high specificity a 170-kDa betagamma conjugate in which we identified residues involved in the intramolecular cross-linking of the 125-kDa beta subunit between its regulatory N terminus and its C terminus. This finding provides an explanation for previously published homodimeric two-hybrid interactions of the beta subunit and suggests a dynamic structural role for the regulatory N terminus of that subunit. The results offer proof of concept for the CrossSearch strategy for analyzing conjugates and are the first to reveal a tertiary structural element of either homologous alpha or beta regulatory subunit of phosphorylase kinase.

Fig. 1.

Flow diagram of the CrossSearch engine. Green rectangles indicate the search engine data input sites that allow entry of the amino acid sequences of the protein (Pr) conjugate pair, chemical cross-linker, protease (e.g. trypsin), and mass lists generated by MS analyses of digests of the conjugate and corresponding non-cross-linked Pr components. Theoretical (Theor.) digests of Pr1 and Pr2 (blue rectangles) may be carried out either by the search engine or uploaded directly from other processing tools, such as PeptideMass from the ExPASy website. Perl programming and MySQL data base iterations are shown in yellow. Orange rectangles denote MySQL tables and matrices that can be manipulated and accessed multiple times throughout the analysis without reiteration of the generating step. *, after contaminant in the experimental column of the search engine designates polyacrylamide and other non-peptide components.

Fig. 2.

Side products of cross-linking. A, Pr Lys ɛ-amines preferentially react with the N-succinimide functional group of GMBS at near-neutral pH to form product 1. Hydrolysis of the maleimide group (product 2) competes either with cross-linking reactions by Pr nucleophiles (product 3) or with exogenous nucleophiles that are present as contaminants or quench reagents to halt cross-linking at specific times (product 4). Conversion of products 3 and 4 to their corresponding maleimic derivatives occurs after hydrolysis and subsequent ring opening of the S-substituted maleimide rings (products 5 and 6). B, targeting of the maleimido functional group is generally favored by Pr Cys thiols (product 7). Hydrolysis of the succinimide group competes with cross-linking reactions by Pr nucleophiles, resulting in monoderivatization of the thiol (product 8). A second mass addition to the thiol may be detected after conversion of the maleimide to the corresponding maleimic derivative (product 9).

Fig. 3.

Cross-linking of PhK with GMBS and detection of a potential cross-linked peptide by FT-ICR MS and search engine analyses of a tryptic digest of the major βγ conjugate. A, PhK (Lane 1) was cross-linked with GMBS (Lane 2) and resolved by SDS-PAGE. Parallel samples were transferred to PVDF membranes and probed with mAbs against all of the subunits. All major conjugates cross-reacted only with anti-β and anti-γ mAbs, not with anti-α or anti-δ (δ = integral calmodulin subunit) mAbs. Cross-linking of PhK by GMBS resulted primarily in the formation of a major conjugate corresponding to a βγ dimer by apparent mass (170 kDa) and cross-reactivity (23). B, the cross-linked βγ dimer was digested in gel with trypsin and analyzed by FT-ICR MS. The monoisotopic peak for a peptide with a signal at m/z 658.3597 (∼10× above background) was further subjected to a tandem MS step, and the resulting spectrum was compared against common contaminants as well as tryptic and non-enzymatic hydrolysis products of all the PhK subunits using the Sequest algorithm (37) included in Bioworks 3.2 (ThermoFinnigan). The absence of significant matches observed for the 658 m/z signal in either the Sequest data sets or mass lists obtained from digests of non-cross-linked β and γ subunit controls (A, Lane 2) together indicated the presence of a potential cross-linked peptide.

Fig. 4.

MS/MS analysis of the signal at m/z 658.3597 identifying a conjugate comprising residues 21–23 and 1037–1042 of the regulatory β subunit of PhK. A, fragmentation pattern of the cross-linked peptide. Small letters denote ions arising from amide cleavages of the peptide backbone and are color-coded with the respective peptide in the conjugate pair (green and black for residues 21–23 and 1037–1042, respectively). B, structure of the GMBS cross-link between Lys-22 and Arg-1040. The capital letters indicate ions resulting from amide cleavage of the reagent and ɛ-amine of Lys-22. C, fragmentation pattern of ions corresponding to cleavage (∼) of the intervening cross-link. D, composition of ions identifying the cross-linked peptide. Intact covalent links formed between peptides are indicated by a colon (:), and covalent links between fragments of the cross-link and peptide are indicated by a centered dot (·). For singly charged ions, it should be noted that one of the two peptide ions covalently attached to either functional group of GMBS is a neutral product of the indicated backbone amide cleavage. * and ^o, loss of ammonia and water.

Fig. 5.

Domain map of the β subunit of PhK showing regions of intramolecular cross-linking by GMBS. Seryl residues within the N-terminal region that are phosphorylated by either cAMP-dependent protein kinase or PhK autophosphorylation are indicated by a P below them. The N-terminal 32 residues of β, indicated by gradation from white to black, represent a region of the subunit that has been shown to regulate homodimeric β interactions in two-hybrid screens (23). A region required for homodimeric interactions of β in two-hybrid screens corresponds to residues 917–1093 (light gray) and includes a stretch of residues (1026–1047) that are a predicted to have high propensity for forming a coiled-coil domain (23). GMBS intramolecular cross-linking of these two regions in the β subunit of the activated PhK complex is indicated by peptides (shown in red lettering) cross-linked through residues Lys-22 and Arg-1040.