Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins

Cellular nucleic acid-binding proteins
(NABPs), namely, DNA-binding
proteins (DBPs) and RNA-binding proteins (RBPs), play important roles
in many biological processes. However, extracting NABPs with high
efficiency in living cells is challenging, which greatly limited their
proteomics analysis and comprehensive characterization. Here, we discovered
that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency
(96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore
developed a Ti4+-IMAC method for the joint extraction of
DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking,
DBPs and RBPs were covalently linked to nucleic acids (NAs) and further
denatured by organic solvents. After Ti4+-IMAC capture,
2000 proteins were identified in 293T cells, among which 417 DBPs
and 999 RBPs were revealed, showing promising selectivity for NABPs.
We further applied the Ti4+-IMAC capture method to lung
cancer cell lines 95C and 95D, which have different tumor progression
abilities. The DNA- and RNA-binding capabilities of many proteins
have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive
characterization of both DBPs and RBPs, which might be utilized to
study their dynamic interactions with nucleic acids.