{Reference Type}: Journal Article
{Title}: Investigating phase separation properties of chromatin-associated proteins using gradient elution of 1,6-hexanediol.
{Author}: Zhu P;Hou C;Liu M;Chen T;Li T;Wang L;
{Journal}: BMC Genomics
{Volume}: 24
{Issue}: 1
{Year}: 2023 Aug 28
{Factor}: 4.547
{DOI}: 10.1186/s12864-023-09600-1
{Abstract}: <B>BACKGROUND: </B>Chromatin-associated phase separation proteins establish various biomolecular condensates via liquid-liquid phase separation (LLPS), which regulates vital biological processes spatially and temporally. However, the widely used methods to characterize phase separation proteins are still based on low-throughput experiments, which consume time and could not be used to explore protein LLPS properties in bulk.<BR><B>RESULTS: </B>By combining gradient 1,6-hexanediol (1,6-HD) elution and quantitative proteomics, we developed chromatin enriching hexanediol separation coupled with liquid chromatography-mass spectrometry (CHS-MS) to explore the LLPS properties of different chromatin-associated proteins (CAPs). First, we found that CAPs were enriched more effectively in the 1,6-HD treatment group than in the isotonic solution treatment group. Further analysis showed that the 1,6-HD treatment group could effectively enrich CAPs prone to LLPS. Finally, we compared the representative proteins eluted by different gradients of 1,6-HD and found that the representative proteins of the 2% 1,6-HD treatment group had the highest percentage of IDRs and LCDs, whereas the 10% 1,6-HD treatment group had the opposite trend.<BR><B>CONCLUSIONS: </B>This study provides a convenient high-throughput experimental method called CHS-MS. This method can efficiently enrich proteins prone to LLPS and can be extended to explore LLPS properties of CAPs in different biological systems.