= Solution
Structural analysis should ask more than whether a candidate was recovered: \b[which surfaces are near one another, which regions change on association, and how are subunits arranged?] Enrichment from part (i) supplies material and candidates, but structural measurements require their own controls.
In <chemical cross-linking mass spectrometry>, react the assembly with a cross-linker of known chemical specificity and spacer properties, digest it and identify linked <peptide> pairs by <tandem mass spectrometry>. An inter-protein link places two reactive residues near one another in at least a sampled conformation; intra-protein links constrain folding or conformation. These constraints can test a structural model or place subunits within an assembly. The useful distance bound includes side chains and linker reach, not only the nominal spacer length. Search-space expansion and ambiguous assignments require error control at the linked-pair level, rather than borrowing an ordinary single-peptide error estimate.
In <hydrogen-deuterium exchange mass spectrometry>, compare the incorporation of <deuterium> into exchangeable backbone positions in free and bound samples over a time course. After quenching and peptide analysis, reduced exchange can reveal protected or stabilized regions. Protection may indicate an interface, but it can also arise from an allosteric change, altered <hydrogen bonds> or reduced structural opening. It therefore constrains a hypothesis rather than identifying a unique atomic contact.
<Native mass spectrometry> can examine intact assemblies and subcomplexes, providing evidence about composition and <stoichiometry>. Gas-phase loss and differing ionization efficiencies must be considered. <Cryo-electron microscopy> can then reveal overall architecture and, with sufficient data quality and suitable particle behaviour, high-resolution interfaces. Heterogeneity, flexible regions and purification artefacts can limit the reconstruction. Combining these observations is often more informative than demanding that one technique answer every structural question.
\b[Quantitative <proteomics> is necessary in both enrichment and structural comparisons.] For an assembled versus disrupted complex, measure component abundances as well as cross-linked-peptide signals. A decreased inter-protein link may reflect loss of one subunit rather than movement of an interface. Comparing cross-link abundance after accounting for recovered protein amounts helps distinguish these explanations. Matched labeling chemistry, reaction time, independent reactions and a dose series reduce artefacts caused by unequal cross-linking or excessive stabilization.
For <hydrogen-deuterium exchange mass spectrometry>, compare matched peptide sequences at multiple labeling times, control back-exchange and retain replicate uncertainty. A consistent local change without a corresponding abundance loss supports a structural effect. Differential <peptide> intensities, exchange measurements and the affinity-enrichment data should be interpreted together; an ordinary protein abundance ratio does not itself measure a distance or a direct interface.
Finally, test a predicted interface by changing selected residues while retaining folding and expression, then repeat the quantitative association assay. Loss of the candidate association with preserved bait integrity strengthens the interface interpretation. \b[The convincing result connects selective recovery, structural constraints and a controlled perturbation], rather than treating one recovered band or one protected segment as a complete structure.
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