Abstract
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.
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Data availability
Raw MS data were deposited to the ProteomeXchange Consortium through the iProX partner repository with dataset identifier PXD021651. The data for CUT&Tag and ATAC-seq were deposited to the National Center for Biotechnology information under BioProject PRJNA1236923. All data are contained within the article and Supplementary Information. Source data are provided with this paper.
Code availability
Code specifically developed for this study is available from GitHub (https://github.com/MonnaHu/Iseq-Kac/tree/master).
Change history
04 June 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41589-025-01955-8
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Acknowledgements
We thank M. Wühr at Princeton University for generously sharing the TMTc+ algorithm. This work was supported by the National Natural Science Foundation of China (82025002 and 32230036 to H.H.; 82394411 and 82170113 to H. Zhang; 81570060, 82073221 and 31870826 to L.D.; 32201216 to Y.G.), National Key R&D Program of China (2022YFA1303200 to L.D.; 2019YFA0110203 to H. Zhang; 2019YFA0110201 to H.H.), National Clinical Research Center for Geriatrics at West China Hospital (Z2024JC002 to L.D.) and West China Hospital 135 project (ZYYC23013 to L.D.; ZYGD22005 to H.H.).
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Authors and Affiliations
Contributions
L.D., H. Zhang and H.H. designed the entire project and wrote the paper. Y.G. and Y.L. performed the MS analysis. H. Zhan, N.W., M.L., Y.X., X.L. and Xiaodong Wang performed the FACS sorting, immunofluorescence staining, confocal microscopy, flow cytometry, immunoblotting, in vitro differentiation, HSC transplantation and CUT&Tag and ATAC-seq experiments. P.G., Y.G. and Y.D. analyzed the sequencing, proteome and acetylome data. Q.P. performed the metabolic analysis of HSCs. Y.G. and S.H. prepared the site-specific polyclonal antibodies. S.H., M.H., W.L. and K.Z. manually checked the MS2 spectra. X.J., Xiuxuan Wang and Z.X. synthesized the chemicals for this study. R.Z., L.G., Xinyuan Wang and Y.L. provided experimental help. P.W., Y.P., P.L., H.X., Y.S. and J.Q. provided helpful suggestions for this project.
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Nature Chemical Biology thanks Hiroaki Honda, Feng-Chun Yang, Hui Ye and Balyn Zaro for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Optimization and evaluation of the Iseq-Kac strategy.
a, Line chart showing the number of total acetylated peptides identified in 50 μg of protein from HCT116 cells labeled with different amounts of Ac-NHS. b, Fan chart depicting the number of mono- and multi-acetylated peptides identified in HCT116 samples labeled with different amounts of Ac-NHS. c, Fan chart showing the number of doubly and other charged peptides generated under different LC-MS/MS conditions. d, Fan chart showing the number of mono- and multi-acetylated doubly charged peptides under varied LC-MS/MS conditions. The C8 condition was used as a standard condition for Iseq-Kac. e, Fan chart showing the number of identified peptides at different mixing ratios, with TMTc cutoff ranging from 0.01 to 0.1. f, False discovery rate (FDR) of acetylated peptide identification at different TMTc cutoffs using a 2:1:1 pre-mixed sample. TMTc ion clusters and MS2 fragments were manually examined. g, Signal-to-noise (S/N) in the low-mass region for 169 identified acetylated peptides, calculated by Proteome Discoverer 2.3. h, Quantification of 2:1:1 pre-mixed digested peptides from HCT116 cells using reporter ions or TMTc. i,j, Quantification of HCT116 peptides pre-mixed at different ratios using reporter ions (i) or TMTc (j). k-n, Peptides from trichostatin A (TSA)-treated HCT116 cells labeled with TMT129 and TMT131 were analyzed by Iseq-Kac. The internal standard (IS) was derived from either different batches of TSA-treated HCT116 cells (k,l), or untreated HCT116 cells and colorectal cancer tissue (m,n). Representative Data from n = 2 (k,m) or n = 3 (g-j) independent experiments. Data are presented as the mean ± s.e.m.
Extended Data Fig. 2 Evaluating Iseq-Kac in low-input samples.
a-h, Peptides from trichostatin A (TSA)-treated HCT116 cells labeled with TMT129 and TMT131 were analyzed by Iseq-Kac. a,b, Venn diagrams showing the overlap of identified (a) and quantified (b) acetylated peptides between two replicates. c, Two-sided Pearson correlation analysis of acetylated peptide ratios (TSA/NC) between replicates. d, Comparison of quantitative results for histone acetylated peptides from Iseq-Kac, IP-MS, and immunoblotting. The immunoblotting data were quantified with ImageJ. e, Histone acetylation sites detected by immunoblotting. Representative western blots from n = 2 or 3 independent experiments. f, Bar graph showing the average number of identified (blue) and quantified (red) acetylated peptides from 100 to 10,000 (per channel) TSA-treated or untreated HCT116 cells. Data are presented as the mean ± s.e.m. of n = 3 independently performed experiments. g, Venn plots showing the overlap of quantified acetylated peptides across three replicates in experiments with 100 to 10,000 cells per channel. h, CV of overlapping acetylated peptides across three replicates using varying numbers of cells. i, Chord diagram showing the classes of proteins identified in different numbers of HCT116 cells. j, Bubble diagram comparing the quantitative results of histone acetylated peptides in different numbers of HCT116 cells. NBT-2015 represents data from Scholz et al.’s work, where acetylome data were obtained using an enrichment-based strategy with 20 mg of protein input.
Extended Data Fig. 3 Evaluation of proteome and acetylome data of HSCs and MPPs.
a, Gating strategy for FACS sorting or analysis of Lin- (CD3e-Ter119-B220-Gr1-CD11b-), HSC (Lin-CD127-Sca1+c-Kit+CD150+CD48-CD34-), CD150hi HSC, CD150lo HSC, MPP1 (Lin-CD127-Sca1+c-Kit+CD150+CD48+), MPP2 (Lin-CD127-Sca1+c-Kit+CD150-CD48+CD135-) and MPP3 (Lin-CD127-Sca1+c-Kit+CD150-CD48+CD135+), GMP (Lin-CD127-Sca1-c-Kit+CD34+FcγRIII/II+), CMP (Lin-CD127-Sca1-c-Kit+CD34+FcγRIII/II-), CLP (Lin-CD127+) and MEP (Lin-CD127-Sca1-c-Kit+CD34-FcγRIII/II-). b-c, Representative data of one of twelve to eighteen independent experiments, for young (b) and aged (c) HSCs and MPPs. d, Flowchart of generating cell-specific Kac libraries for HSCs and MPPs. e, Venn diagrams depicting the overlap of quantified proteins across four replicates in HSCs or MPPs. f, Correlation of quantified proteins among four replicates in HSCs or MPPs. g, Venn diagrams showing the overlap of quantified acetylated peptides among four replicates in HSCs or MPPs. h, Correlation of quantified acetylated peptides among four replicates in HSCs or MPPs.
Extended Data Fig. 4 Proteomes of young and aged HSCs and MPPs.
a, Kinases and phosphatases altered in HSCs or MPPs with age (fold change < 0.85 or > 1.176), ranked by average protein intensity. Red, upregulated in aged cells; blue, downregulated. b, Proteins involved in BCAA, TCA cycle and fatty acid metabolism pathways significantly changed in aged HSCs or MPPs. Red, upregulated in aged cells; blue, downregulated; undetectable proteins labeled as “NA”. c, Relative mRNA expression levels of Foxp1 (top) and Ikzf1 (bottom) in young (blue) and aged (orange) HSCs and MPPs, as determined by qPCR. The cumulative mean values are presented as the fold change normalized to young (n = 3-6 wells, representative data for three independent biological replicates). Data are presented as the mean ± s.e.m. Statistical significance was assessed using two-side t-test (c).
Extended Data Fig. 5 Acetylomes of young and aged HSCs and MPPs.
a, Chord diagram showing the classes of acetylated proteins identified in HSCs and MPPs. b-e, Proteins were categorized into five intervals (R1 to R5) based on average protein abundance ranking, and the proportion of acetylated proteins in each interval was calculated and compared. b, Comparison of protein classes in lowest-abundance proteins intervals (R5), based on our proteome data and Zaro et al.’s proteome data. c, Distribution of acetylated peptides identified by Iseq-Kac across five intervals (R1 to R5), based on protein intensity ranking from our study and Zaro et al.’s study. d,e, Distribution of acetylated peptides identified by classic affinity enrichment-based method across five intervals based on protein intensity ranking. Proteome and acetylome data were obtained from Krug et al.’s study (d) and Yongchao et al.’s study (e). f, Dot blotting assays to confirm the specificities of ALDH2-K430ac and ZNF207-K398ac antibodies using peptides containing acetyllysine (Modified) or unmodified lysine (Unmodified). Each dot contained 10 ng, 100 ng, and 1 µg of peptides, respectively. Representative blots from n = 2 independent experiments.
Extended Data Fig. 6 Age- and metabolism-related acetylome of hematopoietic progenitors.
a, Global ∆As analysis of acetylated proteins in HSCs and MPPs. The acetylated change (∆As) for each protein was calculated as the sum of log2(FC(A/Y)) values of all acetylated peptides of that protein. Dotted lines represent ∆As = ±1. b, Hyper- or hypo-acetylated proteins in aged HSCs or MPPs, with pathway enrichment analysis performed using Metascape (one-sided hypergeometric test). Bubble color indicates the corresponding ∆As values. c, Changes in glycolysis and TCA cycle-related metabolites in HSCs from young and aged mice, determined by mass spectrometry (MS) analysis. Data are presented as the median ± 25% of n = 12 independently performed experiments. Statistical significance was assessed using two-sided two-side t-test. d, Overrepresented NADP metabolic pathways and cellular stress responses with hyper- or hypo-acetylated proteins in aged HSCs or MPPs. Colors represent the ∆As values. Red, hyperacetylated in aged cells; blue, hypoacetylated. NA, acetylation is not detected.
Extended Data Fig. 7 The levels of H4K77ac in young and aged MPPs and LSKs.
a, MS2 spectrum of the peptide containing H4K77ac, with enlarged regions showing the low-mass reporter ion and TMTc ion clusters. b,c, Anti-H4K77ac antibody specificity verified by dot blotting (b) and immunoblotting in TSA-treated and untreated K562 cells (c). Representative blots from n = 2 independent experiments (b,c). d, Cumulative mean values showing the relative mean fluorescence intensities (MFI) of H4K77ac in HSCs and hematopoietic progenitor subsets, as measured by flow cytometry and normalized to young (n = 7-8 mice). e,f, Immunofluorescence of H4K77ac in FACS-sorted young and aged MPPs and LSKs labeled with AF647-conjugated anti-rabbit IgG antibodies. Representative confocal microscopy images of young and aged MPPs and LSKs expressing H4K77ac (red) (e, scale bar, 2μm), with fluorescence quantification of H4K77ac (f) by ImageJ (n = 106-185 cells). The fluorescence intensities were calculated per square pixel and normalized to DAPI. g, Cumulative mean value showing the MFI of H4K77ac in HSPC subpopulations as measured by flow cytometry and normalized to young (n = 6 mice). Data are presented as the mean ± s.e.m. All data are derived from three (d,f) or four (g) independent biological replicates. Statistical significance was assessed via two-way ANOVA followed (d) or two-side t-test (f).
Extended Data Fig. 8 Identification of H4K77ac-related pathways and binding proteins.
a, Fraction of reads falling within peak regions (FRiP, SEACR FDR ≤ 0.05) of H4K77ac CUT&Tag data in young and aged HSCs (n = 20000 HSCs/experiment). b,c, Overlap between genes differentially expressed (downregulated/upregulated) in aged HSCs and genes showing differentially H4K77ac enrichment (TSS ± 3 kb) at their promoters (n = 20000 HSCs/experiment). b, Venn diagrams of overlapping genes. c, Pathway enrichment from Over-Representation Analysis (ORA) of overlapping genes (Fisher’s precision probability tests. p value < 0.01). Blue, downregulated in aged HSCs; Orange, upregulated in aged HSCs. d, Relative mRNA expression levels of signature genes in young (blue) and aged (orange) HSCs, as determined by transcriptome. e, Distribution of ATAC-seq reads (OCRs) at transcription start sites (TSS ± 3 kb) of genes annotated by common peaks (Fig. 4g) associated with lymphocyte differentiation (GO:0030217 for T cell differentiation, GO:0030183 for B cell differentiation) or myeloid cell development (GO:0030099 for myeloid cell differentiation) between young and aged HSCs promoters (n = 20000 HSCs/experiment). f, Distribution of H4K77ac CUT&Tag reads at the transcription start site (TSS ± 3 kb) of genes associated with lymphocytes (GO:0030217 and GO:0030183 gene sets for T and B cell differentiation, respectively) or myeloid cell development (GO:0030099 gene sets for myeloid cell differentiation) in young (left) or aged HSCs (right) promoters (n = 20000 HSCs/experiment). g, Comparative analysis of proteins pulled down by acetylated H4K77 peptide or its unmodified form. h, Immunoblotting showing H4K77ac levels in HCT116 cells treated with HDAC3 inhibitor (RGFP966) or SIRT2 inhibitor (Thiomyristoyl) i, Representative data showing the frequencies of T lymphocytes (CD4-CD8-, CD4+CD8+, CD4+ and CD8+) derived from aged HSCs, determined at 2 weeks post-RGFP966 or DMSO treatment. j, ShRNA-Hdac3-mediated gene knockdown efficiency and H4K77ac levels in K562 cells, assessed by immunoblotting. Data are presented as the median ± 25% (a,e,f) or mean ± s.e.m. (d). All data are derived from three independently biological replicates (a,d,e,f). Representative blots from three independent experiments (h,j). Statistical significance was assessed using the two-side Wilcoxon rank sum test (d) or two-side Kruskal-Wallis rank sum test (e,f).
Extended Data Fig. 9 H4K77ac specifically regulated by the deacetylase HDAC3.
a-j, In vitro differentiation assay of aged HSCs infected with shRNA-Hdac3 (shRNA#1) or vector control. Representative data (a,c,f,h) and cumulative mean values (b,d,e,g,i,j) of T lymphocyte (CD4-CD8-, CD4+CD8+), B lymphocyte (CD19+), myeloid cell (CD11b+) frequencies and H4K77ac abundance in CD45+mCherry+ cells derived from shRNA-Hdac3-infected HSCs or vector control infected HSCs. The cumulative mean values are presented as the fold change normalized to control (n = 14-20 wells). k-m, Representative data (k,m) and cumulative mean values (l,n) of H4K77ac, H3K27ac and H3K9/K14ac in RGFP966-treated (1/5/10 µM) or DMSO-treated HSCs (n = 3 mice). o, Pre-ranked GSEA of genes enriched by H4K77ac in RGFP966-treated versus DMSO-treated HSCs from young mice. Differential peaks (p value < 0.01) were identified using DiffBind in R. p-r, Comparisons between genes co-regulated by H4K77ac and H3K27ac. p, Venn diagrams showing the numbers of overlapping genes. q, Volcano plot displaying the genes differentially expressed between aged and young HSCs based on the 304 overlapping genes. Dotted lines indicate Log2FC = ± log2(1.5), p value adjusted ≤ 0.05. Blue indicates downregulated genes in aged HSCs, orange indicates upregulated genes in aged HSCs, and gray indicates no significant difference. r, Bubble plot showing the pathway enrichment results of the 304 genes, based on GSEA. NES, normalized enrichment score. s,t, Representative immunoblotting showing the expression of H4 (s) or H3 (t) in Lin- bone marrow cells infected with H4K77 (s) or H3K27 (t) mutant virus or vector control. u,v, Cumulative mean value showing the MFI of GFP in GFP+CD45+ cells derived from HSCs ectopically expressing H4K77R/Q (u) or H3K27R/Q (v) (n = 5-8 wells for H4K77 mutations; n = 4-6 wells for H3K27 mutations). All data are derived from three independently biological replicates (a-n and u-v). Representative blots from n = 2 or 3 independent experiments (s,t). Data are presented as the mean ± s.e.m. Statistical significance was assessed using two-side t-test (b,d,e,g,i,j), two-way ANOVA followed by Tukey’s multiple comparisons adjustments (l,n), two-side Wilcoxon Rank Sum Tests (q) or two-side Permutation test (o,r).
Supplementary information
Supplementary Information
Supplementary Figs. 1–3 and legends for Supplementary Tables 1–6.
Supplementary Table 1
Optimization and evaluation of the Iseq-Kac strategy.
Supplementary Table 2
Acetylated peptides or proteins identified and quantified in the aging-associated proteome study of HSC and MPPs.
Supplementary Table 3
The parameters used in MRM for targeted validation of acetylated peptides.
Supplementary Table 4
List of H4K77ac-enriched genes in young and aged HSCs, identified by CUT&Tag assay.
Supplementary Table 5
Acetylated peptides quantified in RGFP966-treated and untreated HSCs.
Supplementary Table 6
Genes enriched with H4K77ac peaks in RGFP966-treated and DMSO-treated HSCs, identified by CUT&Tag assay.
Supplementary Data 1
The expression of H4 in young and aged MPPs and LSKs.
Supplementary Data 2
H4K77ac but not H3K27ac regulates differentiation potential of HSCs.
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Gong, Y., Zhan, H., Wei, N. et al. Acetylation profiling by Iseq-Kac reveals insights into HSC aging and lineage decision. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01916-1
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DOI: https://doi.org/10.1038/s41589-025-01916-1
