Acetylation profiling by Iseq-Kac reveals insights into HSC aging and lineage decision

Profiling post-translational modifications face challenges with low-input samples. We developed Iseq-Kac (internal standard-assisted enrichment-free approach for high-throughput quantitative analysis of lysine acetylation) to profile the acetylome in as few as 10³-10⁴ cells. By using a hyperacetylated internal standard, Iseq-Kac can be used in mass spectrometry (MS) to enhance MS1 signals and facilitate MS2 fragmentation of acetylated peptides. Using Iseq-Kac, we quantified 675-1,471 acetylated peptides per analysis from 10⁴ hematopoietic stem cells (HSCs) or multipotent progenitors. Validation by targeted MS, site-specific antibodies and functional assays linked aging-related proteome and acetylome changes to HSC lineage decision. A pronounced decrease in acetylation at H4 lysine 77 (H4K77ac) was observed in aged HSCs, linked to histone deacetylase 3 (HDAC3) activity. HDAC3 inhibition or knockdown in HSCs significantly promoted lymphocyte differentiation. Mimicking H4K77ac through H4K77Q expression enhanced B cell differentiation while repressing myeloid differentiation. Overall, Iseq-Kac enables robust low-input acetylome profiling and reveals epigenetic mechanisms underlying lineage skewing in aged HSCs.