Profiling Protein–Protein Interactions in the Human Brain by Refined Cofractionation Mass Spectrometry

Proteins usually execute their biological functions through
interactions
with other proteins and by forming macromolecular complexes, but global
profiling of protein complexes directly from human tissue samples
has been limited. In this study, we utilized cofractionation mass
spectrometry (CF-MS) to map protein complexes within the postmortem
human brain with experimental replicates. First, we used concatenated
anion and cation Ion Exchange Chromatography (IEX) to separate native
protein complexes in 192 fractions and then proceeded with Data-Independent
Acquisition (DIA) mass spectrometry to analyze the proteins in each
fraction, quantifying a total of 4,804 proteins with 3,260 overlapping
in both replicates. We improved the DIA’s quantitative accuracy
by implementing a constant amount of bovine serum albumin (BSA) in
each fraction as an internal standard. Next, advanced computational
pipelines, which integrate both a database-based complex analysis
and an unbiased protein–protein interaction (PPI) search, were
applied to identify protein complexes and construct protein–protein
interaction networks in the human brain. Our study led to the identification
of 486 protein complexes and 10054 binary protein–protein interactions,
which represents the first global profiling of human brain PPIs using
CF-MS. Overall, this study offers a resource and tool for a wide range
of human brain research, including the identification of disease-specific
protein complexes in the future.