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 userfriendly search engine, CrossSearch, that provides the foundation for an overarching strategy to detect crosslinked peptides from the digests of large (>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 (␣␤␥␦) 4 phosphorylase kinase model complex was cross-linked to form with high specificity a 170-kDa ␤␥ conjugate in which we identified residues involved in the intramolecular cross-linking of the 125-kDa ␤ subunit between its regulatory N terminus and its C terminus. This finding provides an explanation for previously published homodimeric two-hybrid interactions of the ␤ 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 ␣ or ␤ regulatory subunit of phosphorylase kinase. Molecular & Cellular Proteomics 7:739 -749, 2008.Chemical cross-linking of proteins is a versatile technique with uses ranging from screening to obtaining relative or absolute structural information for interacting proteins (1). For large proteins that are refractive to techniques such as crystallography or NMR, cross-linking provides a means of gaining moderate to low resolution structural data by generating distance measurements between specific regions of interacting proteins (intermolecular cross-linking) or individual proteins (intramolecular cross-linking). Advances in modern MS methods have promoted a resurgence in the use of crosslinking by eliminating the need for large quantities of conjugates (term used herein to denote either proteins or peptides that are covalently cross-linked) to isolate and identify distinct regions of inter-or intramolecular cross-linked peptides (2, 3). Nanomolar amounts of conjugates are now routinely digested in gel to provide sufficient quantities of peptides for detection by MS meth...

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

“…We assume that cross-linking of the ␤ subunit in the intact complex captures a conformation of this subunit that is induced by its interactions with all subunits, including itself, in the 4°structure of PhK. In further support of the use of this cross-linking constraint, we recently demonstrated that PhK binds the glucoamylase inhibitor acarbose and that this binding perturbs intramolecular cross-linking of the ␤ subunit by GMBS (64), which cross-links the GHL domain to the C terminus of the ␤ subunit (30). The direct binding of acarbose by the ␤ GHL domains would allow a potential mechanism for inducing a conformational change in this subunit through altering potential interactions between the predicted ␤ GHL and the HRL domains, consistent with results from the intramolecular cross-linking of ␤ by DFDNB.…”

“…The corresponding log file was used to generate a list of parent ions for which the corresponding charges and tandem mass spectra were obtained. A list of potential conjugates was generated from the resulting mass list ((M ϩ H) ϩ ) by previously described methods (30), and the fragmentation patterns of all viable candidates were analyzed for consistency with the predicted chemistry of cross-linking (30).…”

“…We report nearly full coverage of the subunit by threading with templates corresponding to GH-15 family members and the protein phosphatase 2A (PP2A) subunit PR65/A (PP2AA). We directly demonstrate for the intact complex by top-down MS and chemical cross-linking that the ␤ subunits compose the central bridge region of PhK and that rigid-body docking of their theoretical counterparts in the individual bridges of the native PhK cryo-EM envelope correlates well with the known structural details for this subunit in the complex (7,16,30).…”

“…Within the (␣␤␥␦) 4 PhK complex, the structure of ␤ is undoubtedly influenced by subunit interactions with itself and with the other three subunits, as evidenced by chemical cross-linking (7,29,30,38,54), EM three-dimensional reconstructions (10), and partial proteolysis (26). As opposed to the ␣, ␥, and ␦ subunits, which have been isolated successfully either individually (8,55,56) or in subcomplexes of PhK (57,58), the ␤ subunit appears to be stable or isolable only in the context of the intact (␣␤␥␦) 4 complex.…”

“…To screen for potential intramolecular conjugates, the ␤ monomeric band remaining after treatment with DFDNB was digested in-gel with trypsin, and the resultant peptides were resolved and measured by Fourier transform ion cyclotron resonance MS. The mass list for the digest was then evaluated using the CrossSearch method (30). After culling experimental masses corresponding to those predicted either for nonmodified peptides or for side products of chemical cross-linking, a mass (m/z Theor ϭ 2297.207) predicted for a peptide corresponding to cross-linking between regions of the N terminus (residues 48 -56, LDYYYKIVK) and C terminus of ␤ (residues 1021-1028, VDLDKLVK) matched that of a peptide mass (m/z Exp ϭ 2297.197; 4.37 ppm error) measured experimentally.…”