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.