Two-Dimensional Fractionation Method for Proteome-Wide Cross-Linking Mass Spectrometry Analysis

Cross-linking
mass spectrometry (XL-MS) is an emergent technology
for studying protein–protein interactions (PPIs) and elucidating
architectures of protein complexes. The development of various MS-cleavable
cross-linkers has facilitated the identification of cross-linked peptides,
enabling XL-MS studies at the systems level. However, the scope and
depth of cellular networks revealed by current XL-MS technologies
remain limited. Due to the inherently broad dynamic range and complexity
of proteomes, interference from highly abundant proteins impedes the
identification of low-abundance cross-linked peptides in complex samples.
Thus, peptide enrichment prior to MS analysis is necessary to enhance
cross-link identification for proteome-wide studies. Although chromatographic
techniques including size exclusion (SEC) and strong cation exchange
(SCX) have been successful in isolating cross-linked peptides, new
fractionation methods are still needed to further improve the depth
of PPI mapping. Here, we present a two-dimensional (2D) separation
strategy by integrating peptide SEC with tip-based high pH reverse-phase
(HpHt) fractionation to expand the coverage of proteome-wide XL-MS
analyses. Combined with the MS-cleavable cross-linker DSSO, we have
successfully mapped in vitro PPIs from HEK293 cell
lysates with improved identification of cross-linked peptides compared
to existing approaches. The method developed here is effective and
can be generalized for cross-linking studies of complex samples.