Proximity-labeling proteomics (APEX2 peroxidase or BioID/TurboID biotin ligase) fuses an enzyme "bait" to a protein of known location. A short pulse generates reactive biotin radicals or biotin-AMP that diffuse only a few tens of nanometres before they are quenched or hydrolysed, covalently tagging nearby "prey" proteins. Mass spectrometry then identifies which proteins were biotinylated, revealing the local proteome. Imaging mass spectrometry instead rasters a probe across a tissue section, collecting a spectrum per pixel to map molecules spatially, while multiplexed immunofluorescence stains several protein channels at once to cross-validate localization.
Labeling probability rises with pulse time and falls off with distance from the bait, modeled here as a pseudo-first-order kinetic process with an exponential distance-decay rate constant:
P_label(d, t) = 1 − exp[ −k · exp(−d/λ) · t ]
d = distance from bait enzyme (nm)
λ = characteristic reactive radius (APEX2 ≈ 20 nm, BioID ≈ 10 nm)
k = pulse intensity (H2O2/biotin exposure)
t = cumulative reaction time (s)
Each protein instance carries a fixed random threshold; once P_label exceeds it the tag is irreversible (biotinylation does not reverse), so labeling accumulates and saturates over time exactly as in a real pulldown.
- Reactive radius λ — sets how far the radical diffuses before quenching; smaller λ gives higher spatial resolution but fewer hits.
- Protein instances — scene population, used to compute local protein density.
- Pulse intensity k — how aggressive the labeling chemistry is (longer/stronger H2O2 or biotin pulse).
- Fixed Bait / Raster (Imaging MS) — bait stays at one organelle (classic proximity labeling) or sweeps a scan path across the cell surface, mimicking a rastering imaging-MS probe collecting pixel-by-pixel spectra.
- CH1–CH4 — toggle visibility of four simulated multiplexed-IF marker channels without altering the underlying kinetics.
- Reset Reaction — clears cumulative labeling and reassigns random thresholds, restarting the pulldown.
This combination — proximity labeling for interaction neighborhoods, imaging MS for spatial ion maps, and multiplex IF for orthogonal validation — is exactly how modern spatial proteomics pipelines triangulate where proteins actually sit inside cells and tissues.