Past exam of the systems biology course of the University of Cambridge / 2024 / Paper 2 / 3

Affinity purification and proximity labeling answer related but different questions. The former enriches material that remains associated during extraction; the latter marks a neighbourhood before extraction. Neither alone proves a direct protein-protein interaction.

In affinity purification-mass spectrometry, isolate a bait protein with an antibody or an endogenous epitope tag. Lyse under conditions chosen to preserve the relevant protein complex, capture the bait on an affinity matrix, wash away background and identify co-purifying proteins by liquid chromatography-mass spectrometry after digestion into peptides. Co-immunoprecipitation is the antibody-based version of this association test. Gentle conditions preserve more weak interactions but also increase background; stringent washing improves specificity at the cost of labile partners. Extraction can disrupt membrane assemblies or create associations that were not present in intact cells.

Controls should include matched tag-only or untagged cells, an appropriate antibody control, input abundance and, where feasible, a binding-defective bait that retains its normal localization. Check that tagging has not changed the bait's abundance, localization or function. Reciprocal capture of a candidate partner is useful validation. If an association might be bridged by RNA or DNA, compare nuclease-treated and untreated samples while recognizing that nuclease treatment may dismantle a biologically genuine assembly.

For proximity labeling, fuse the bait to TurboID, which activates biotin and covalently labels accessible nearby proteins in living cells. After a timed labeling pulse, harsh lysis can be used because recovery no longer requires the original assembly to survive. Capture biotinylated material using streptavidin and identify enriched proteins by mass spectrometry. This can recover transient or insoluble neighbourhoods missed by ordinary extraction. A localization-matched control enzyme, no-added-biotin condition and matched expression help separate the bait's neighbourhood from diffuse labeling and endogenous biotinylated proteins.^[ref]

The label records proximity, accessibility and time spent nearby. A neighbour need not bind the bait; a real binder might label poorly if reactive groups are inaccessible. Pulse duration therefore trades temporal selectivity against labeling yield, rather than defining an exact universal distance cutoff.

Quantification distinguishes enrichment from a list of contaminants. Use label-free quantification of peptide signals across independent bait and control purifications, or SILAC in compatible cell cultures to compare light and heavy samples. In SILAC, isotope-containing amino acids enter newly synthesized proteins; matched peptides form distinguishable mass pairs. A label swap helps identify isotope-condition confounding. Tandem mass tags offer multiplexed comparison using reporter signals, but interfering co-isolated peptides can compress ratios.

Estimate enrichment and its reproducibility relative to matched controls, accounting for bait recovery and input abundance. Common abundant contaminants should not be promoted solely because many spectra identify them. Statistical scoring such as SAINT models the contrast between specific and background recovery across controls and replicates, allowing candidate associations to be ranked with estimated error rates.^[ref] A reproducibly enriched candidate is evidence for the assay-defined association or neighbourhood, not automatically a direct binding partner or fixed stoichiometric subunit.

Structural analysis should ask more than whether a candidate was recovered: 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.

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. 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.