Cross-linking Mass Spectrometry Analysis of the Yeast Nucleus Reveals Extensive Protein–Protein Interactions Not Detected by Systematic Two-Hybrid or Affinity Purification-Mass Spectrometry

Saccharomyces cerevisiae has the most comprehensively
characterized protein–protein interaction network, or interactome,
of any eukaryote. This has predominantly been generated through multiple,
systematic studies of protein–protein interactions by two-hybrid
techniques and of affinity-purified protein complexes. A pressing
question is to understand how large-scale cross-linking mass spectrometry
(XL-MS) can confirm and extend this interactome. Here, intact yeast
nuclei were subject to cross-linking with disuccinimidyl sulfoxide
(DSSO) and analyzed using hybrid MS2-MS3 methods. XlinkX identified
a total of 2,052 unique residue pair cross-links at 1% FDR. Intraprotein
cross-links were found to provide extensive structural constraint
data, with almost all intralinks that mapped to known structures and
slightly fewer of those mapping to homology models being within 30
Å. Intralinks provided structural information for a further 366
proteins. A method for optimizing interprotein cross-link score cut-offs
was developed, through use of extensive known yeast interactions.
Its application led to a high confidence, yeast nuclear interactome.
Strikingly, almost half of the interactions were not previously detected
by two-hybrid or AP-MS techniques. Multiple lines of evidence existed
for many such interactions, whether through literature or ortholog
interaction data, through multiple unique interlinks between proteins,
and/or through replicates. We conclude that XL-MS is a powerful means
to measure interactions, that complements two-hybrid and affinity-purification
techniques.