A Proteomics Strategy for the Identification of FAT10-Modified Sites by Mass Spectrometry

The
ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10)
is uniquely expressed in mammals. The fat10 gene
is encoded in the MHC class I locus in the human genome and is related
to some specific processes, such as apoptosis, immune response, and
cancer. However, biological knowledge of FAT10 is limited, owing to
the lack of identification of its conjugates. FAT10 covalently modifies
proteins in eukaryotes, but only a few substrates of FAT10 have been
reported until now, and no FATylated sites have been identified. Here,
we report the proteome-scale identification of FATylated proteins
by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
We identified 175 proteins with high confidence as FATylated candidates.
A total of 13 modified sites were identified for the first time by
a modified search of the raw MS data. The modified sites were highly
enriched with hydrophilic amino acids. Furthermore, the FATylation
processes of hnRNP C2, PCNA, and PDIA3 were verified by a coimmunoprecipitation
assay. We confirmed that most of the substrates were covalently attached
to a FAT10 monomer. The functional distribution of the FAT10 targets
suggests that FAT10 participates in various biological processes,
such as translation, protein folding, RNA processing, and macromolecular
complex assembly. These results should be very useful for investigating
the biological functions of FAT10.