Abdulsalam, S. | bioRxiv Science

bioRxiv2026

Extraction-dependent bone proteomics reveals distinct stable and dynamic protein modules during early post-exposure degradation

Bone is a highly durable biological tissue widely used in forensic, archaeological, and anthropological investigations; however, efficient protein recovery and understanding of protein stability over time remain major challenges in skeletal proteomics. Here, we systematically evaluated three bone protein extraction workflows and integrated them with data-independent acquisition (DIA) mass spectrometry to assess proteome coverage, reproducibility, and temporal protein dynamics under environmentally exposed conditions. Comparative analysis demonstrated that extraction strategy is a primary determinant of detectable proteome composition. EDTA-based demineralization followed by SDS extraction provided the deepest proteome coverage and highest reproducibility, whereas guanidine hydrochloride extraction preferentially enriched collagen and extracellular matrix proteins. In contrast, acid-based extraction yielded limited protein recovery. Temporal profiling of bone samples collected at 10 and 45 days post-exposure revealed two distinct protein classes. A temporally stable module, enriched in collagens and extracellular matrix proteins including COL1A2, COL5A2, BGN, SPARCL1, and NID2, exhibited minimal abundance change, indicating resistance to environmental degradation. In contrast, temporally dynamic proteins, enriched in mitochondrial, metabolic, and intracellular pathways such as ACO2, OGDH, PDHA1, ATP5PO, and PFKM, showed marked decline over time. These findings support a two-compartment model of bone protein preservation in which matrix-embedded proteins are preferentially retained while exposed intracellular proteins undergo progressive degradation. Collectively, this study establishes an integrated framework linking extraction methodology with temporal proteome stability and identifies candidate markers for skeletal preservation assessment and temporal biomarker development in forensic and archaeological applications.

systems biology↗

bioRxiv2026

Inhibition of Dot1L Histone Methyltransferase Expands Bone Injury-Responsive CXCL12⁺ Stromal Progenitors

Adult bone marrow contains a heterogeneous network of skeletal stromal and progenitor cells (SSPC) that maintain bone homeostasis, support the hematopoietic niche, and drive the regenerative responses to injury. Despite their central role in bone regeneration, the mechanisms that regulate injury-induced SSPC activation and fate transitions remain incompletely understood. Here we identify Disruptor of telomeric silencing 1 like (DOT1L), the sole histone methyltransferase responsible for H3K79 methylation, as a critical regulator that restrains stromal activation. Using complementary genetic, pharmacologic, single-cell transcriptomic and injury models, we show that either Prrx1 lineage Dot1L haploinsufficiency or acute pharmacological Dot1L inhibition promote SSPC expansion, but through distinct programs: haploinsufficiency drives the expansion of Cxcl12+ CAR cells, whereas acute pharmacologic inhibition enriches fibroblastic-like stromal states. Single Cell Regulatory Network Inference and Clustering (SCENIC) analysis identifies DOT1L as a stabilizer of homeostatic marrow-supportive transcriptional programs, whose disruption facilitates transition to injury responsive stromal states. Partial loss of Dot1L in the Prrx1 lineage enhances injury-induced intramedullary mineralization in vivo. Collectively, these findings establish DOT1L as a gatekeeper of the stromal progenitor state that restrains lineage commitment and restrains the magnitude of the SSPC response following injury.

cell biology↗