1. The original intention of PhaSepDB 2.0

Phase separation (PS) regulates various biological processes, such as the assembly of membraneless organelles (MLOs), signaling transduction, transcription regulation, protein degeneration et al. Various proteins have been proven to undergo PS, this property is important for many proteins to execute their functions, and aberrant PS may lead to diseases.
In 2019/10/04, we presented PhaSepDB which provided experimental verified PS proteins and MLO related proteins. PhaSepDB is well-received to the PS community, with more than 700 unique visitors per day.
Numerous works in this field have been published since then, thus we update to PhaSepDB 2.0, which contains 961 PS entries, 698 low throughput MLO related entries and 6981 high throughput MLO related entries, we also provide more detailed annotation for all entries.

2. PS entries collection and classification

We rechecked literatures in PhaSepDB 1.0 and filtered PubMed search results from 2019/07/01 (endpoint of the first version) to 2021/05/01 with keywords:
“(phase transition[Title/Abstract] OR phase separation[Title/Abstract] OR membraneless organelles[Title/Abstract] OR biomolecular condensates[Title/Abstract]) AND protein) AND cell”
961 PS entries for 593 proteins were extracted from these literatures, and 280 entries for 187 proteins came from the first version. As a comparation, there are 121 proteins in PhaSePro (10.1093/nar/gkz848) and 216 natural proteins in LLPSDB (10.1093/nar/gkz778). These entries were classified into 356 “PS-self” and 605 “PS-other” entries, the former means that the protein can undergo PS in vitro by itself, while the latter means the protein require other partners or nucleic acids to form biomolecular droplet in vitro or can form or be recruited to biomolecular droplet in vivo.

3. PS entries annotation

For PS entries, each entry was assigned with one or more of the four evidences based on original publication.
1) “in vitro droplet formation”, 2) “In vivo droplet formation”: the protein can form or be recruited in biomolecular droplet in vitro or in vivo.
3) “In vitro FRAP”, 4) “In vivo FRAP”: Fluorescence recovery after photobleaching (FRAP) experiments show the mobility of proteins within the droplet in vitro or in vivo.
We enriched and reformatted the previous annotation for all entries to include:
1. 787 material states of the PS droplets (liquid, hydrogel, solid).
2. 672 regions used in PS experiments, and 511 domains which are important for the proteins to undergo PS.
3. The PS partners for the protein, including 531 proteins, 147 RNAs and 84 others (such as chemicals and DNAs).
4. The regulation of the protein’s PS ability, including 113 posttranslational modifications (PTMs), 266 mutations, 80 oligomerizations, 26 repeats and 4 alternative splicings.

4. MLO and MLO related entries collection

Besides the 961 entries with clear phase separation evidence, PhaSepDB 2.0 contains 698 low-throughput MLO related entries as well (96 entries from PhaSepDB 1.0 Reviewed data and 411 entries from PhaSepDB 1.0 Database reviewed data). These entries refer to proteins localize in MLOs with in vivo experiments, but the original literature didn’t mention phase separation. Furthermore, we collected 6981 high-throughput MLO related entries from 17 papers, these papers were listed below.
For users interested in specific MLO, we provide navigation that enable users to browse based on specific MLO location. Currently, information for 59 MLOs were gathered from dispersive literatures, including both classic and newly discovered bodies.