A Constitutional Isomer Selective Chemical Proteomic Strategy for System-wide Profiling of Protein Lysine 5-Hydroxylation

Lysine 5-hydroxylation (5-Hyl) has been well recognized as an essential protein post-translational modification regulating cellular structural stability, RNA alternative splicing and epigenetic gene expression. System-wide enrichment and quantification of 5-Hyl targets have been challenging due to their chemical inert nature and difficulties in differentiating structural isomers in a complex biological sample. Here, we report the development of an efficient chemical proteomic workflow for affinity enrichment and constitutional isomer specific profiling of endogenous 5-Hyl substrates based on highly selective periodate chemistry. Our study confidently identified over 1600 5-Hyl sites on 630 proteins in human 293T cells, revealing functional significance of the modification in protein structure, transcription and chromatin regulation. Analysis of histone 5-Hyl sites showed that histones H2B and H1 are major targets of the 5-hydroxylysine epigenetic mark. Quantitative proteomic analysis through our chemical enrichment workflow identified specific 5-Hyl substrate proteins mediated by the overexpression of Jumonji-domain containing protein 6 (JMJD6). Our study uncovered two cancer-relevant alternative splice isoforms of JMJD6 that regulate 5-Hyl proteins in distinct cellular pathways, providing unique insights into the functional roles of JMJD6 alternative splicing in transcriptional regulation and cellular development.