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Cross-linking/Mass spectrometry (XLMS) is a consolidated technique for structural characterization of proteins and protein complexes. Despite its success, the cross-linking chemistry currently used is mostly based on N-hydroxysuccinimide (NHS) esters, which react primarily with lysine residues. One way to expand the current applicability of XLMS into several new areas is to increase the number of cross-links obtainable for a target protein. We introduce a multiplex chemistry (denoted XPlex) that targets Asp, Glu, Lys, and Ser residues. XPlex can generate significantly more cross-links with reactions occurring at lower temperatures and enables targeting proteins that are not possible with NHS ester-based cross-linkers. We demonstrate the effectiveness of our approach in model proteins as well as a target Lys-poor protein, SalBIII. Identification of XPlex spectra requires a search engine capable of simultaneously considering multiple cross-linkers on the same run; to achieve this, we updated the SIM-XL search algorithm with a search mode tailored toward XPlex. In summary, we present a complete chemistry/computational solution for significantly increasing the number of possible distance constraints by mass spectrometry experiments, and thus, we are convinced that XPlex poses as a real complementary approach for structural proteomics studies.

A series of bisaryl-substituted fluorescent maleimides was synthesized via the Heck arylation. The compounds showed broad fluorescence emission bands in the visible region, a large Stokes shift in polar solvents and emission quantum yields varying from 0.04 to 0.71, depending on the structure and solvent medium. The difference in dipole moments of ground and excited electronic states of about 12 Debye is ascribed to a substantial charge shift and push-pull character of bisaryl-substituted maleimides. The fluorescence decays of N-benzyl-3,4-bis(4-methoxyphenyl)-1H-pyrrole-2,5-dione (compound 5a) are biexponential with short (1.3-7.6 ns) and long lived (11.5-13.6 ns) components in polar solvents, but in 1,4-dioxane and THF the decays become single exponential. On the other hand, N-benzyl-3-(4-methoxyphenyl)-4-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione (compound 5b) exhibited a biexponential decay in DMSO and in DMF with much shorter decay components, and such behavior indicated a charge shift process combined with solvent assisted proton transfer in the excited state.

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