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.
Highlights
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
Using the MSEC approach with GMBS, we demonstrate that this reagent cross-links the hexadecameric phosphorylase kinase (PhK) complex to form sufficient amounts of a ␥ dimer for analysis and that the formation of this conjugate occurs with a tolerable amount of monoderivatized products
Using this strategy in combination with FT-ICR MS, we identified residues involved in the intramolecular crosslinking of the 125-kDa regulatory  subunit of PhK in the context of the entire hexadecameric complex
Summary
PhK, phosphorylase kinase; GMBS, N-[␥-maleimidobutyryloxy]succinimide ester; MSEC, minimal stoichiometric excess of cross-linker; mAb, monoclonal antibody; NDC, nuclear division cycle; Pr, protein; LC ESI, liquid chromatography electrospray ionization; PHP, hypertext preprocessor; theor., theoretical; XHTML, extensible hypertext markup language. To minimize the number of candidate ions observed in the analysis of large proteins by chemical cross-linking, we have used an alternative approach to the labeling strategies by using a minimal stoichiometric excess of cross-linker, which for convenience we term MSEC. The methods described above streamline somewhat the analysis of cross-linked proteins, predicting the chemical composition of any specific candidate ion requires considerable computational power given the large pool of products possible from the cross-linking and digestion of conjugates. CrossSearch provides the underpinning for an overall strategy for predicting and verifying the composition of cross-linked peptides from both small and large conjugates, especially when used in conjunction with MSEC Using this strategy in combination with FT-ICR MS, we identified residues involved in the intramolecular crosslinking of the 125-kDa regulatory  subunit of PhK in the context of the entire hexadecameric complex. This crosslinking result represents the first report of a tertiary structural element for either of the two large, homologous ␣ and  subunits of PhK
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