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<p>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 10<sup>3</sup>–10<sup>4</sup> 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 10<sup>4</sup> 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.</p><p></p>

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

  • 期刊论文

  • 发表日期：

    2025年5月26日

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  • Yanqiu Gong,
  • Huiwen Zhan,
  • Ni Wei,
  • Min Liu,
  • Yu Liu,
  • Pengbo Guan,
  • Yusi Xie,
  • Yujun Deng,
  • Qianlun Pu,
  • Xiaoxian Lou,
  • Xiaodong Wang,
  • Rou Zhang,
  • Ping Wang,
  • Xiuxiu Jin,
  • Xiuxuan Wang,
  • Zhiqiang Xu,
  • Li Gao,
  • Xinyuan Wang,
  • Siyu He,
  • Ying Lu,
  • Meng Hu,
  • Wanmeng Li,
  • Kun Zheng,
  • Yong Peng,
  • Peng Lei,
  • Heng Xu,
  • Yujun Shi,
  • Jun Qin,
  • Hongbo Hu,
  • Huiyuan Zhang,
  • Lunzhi Dai

摘要

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.