Combining Proximity Labeling and Cross-Linking Mass Spectrometry for Proteomic Dissection of Nuclear Envelope Interactome

Proximity labeling (PL) and chemical
cross-linking (XL) mass spectrometry
are two powerful methods to dissect protein–protein interactions
(PPIs) in cells. Although PL typically captures neighboring proteins
within a range of 10–20 nm of a single bait protein, chemical
XL defines direct protein–protein contacts within 1 nm in a
systemic manner. Here, we develop a new method, named PL/XL-MS, to
harness the advantages of both PL and XL. PL/XL-MS can enrich a subcellular
compartment by PL and simultaneously identify PPIs of multiple proteins
from XL data. We applied PL/XL-MS to dissect the human nuclear envelope
interactome. PL/XL-MS successfully enriched the nuclear envelope proteins
and identified most known inner nuclear membrane proteins. By searching
the cross-linked peptides, we successfully observed 109 literature-curated
PPIs of 14 nuclear envelope proteins. Based on the homoprotein XL
data, we experimentally characterized a nuclear matrix protein, Matrin-3,
and observed its preferential localization near the nuclear envelope.
PL/XL-MS is a simple and general method for studying protein networks
in a subproteome of interest.