Acetylomics Analysis Services — Proteome-Wide Lysine and N-Terminal Acetylation Profiling

Histone Acetylation & Epigenetic Regulation

Acetylation of histone H3 (K9ac, K14ac, K18ac, K27ac, K36ac, K56ac) and H4 (K5ac, K8ac, K12ac, K16ac) marks open, transcriptionally active chromatin regions. H3K27ac is a superenhancer mark; H3K9ac marks active promoters; H4K16ac disrupts higher-order chromatin compaction. These marks are installed by p300/CBP, PCAF, GCN5, and related HATs, and erased by HDAC1–3 (class I, nuclear) and HDAC4/5/7/9 (class IIa). Quantitative acetylomics of histone marks is the primary analytical readout for HDAC inhibitor treatment, HAT activator studies, and epigenetic drug MoA characterization.

Non-Histone Protein Acetylation & Signaling

p53 is acetylated at K382 by p300 in response to DNA damage — increasing its transcriptional activity and apoptotic function; HDAC inhibition raises p53 K382 acetylation ~4-fold. NF-κB RelA is acetylated at K310 by p300/CBP upon activation — augmenting DNA binding and priming ubiquitin-dependent clearance. PCNA acetylation at K164 promotes non-template repair polymerase recruitment. Tubulin acetylation at K40 by αTAT1 marks stable microtubule populations targeted by HDAC6. These and thousands of other non-histone acetylation events are detectable and quantifiable by acetylomics — the full scale of which cannot be captured by any antibody-based single-site assay.

Metabolic Acetylation & Mitochondrial Regulation

Mitochondrial proteins are extensively acetylated — driven partly by the high local concentration of acetyl-CoA in the mitochondrial matrix, which enables nonenzymatic acetylation at susceptible lysines. SIRT3 is the primary mitochondrial deacetylase, deacetylating and activating key TCA cycle and fatty acid oxidation enzymes. Acetylomics of mitochondria-enriched fractions or whole-cell lysates identifies SIRT3 substrate networks, metabolic enzyme regulation, and the mitochondrial response to nutrient state changes — connecting protein acetylation directly to metabolic flux and energy homeostasis. Acetyl-CoA availability links acetylomics directly to metabolomics and nutrient-sensing research.

Acetylation Stoichiometry — Why It Matters

Most lysine acetylation occurs at very low stoichiometry. Proteome-scale studies have shown that the median acetylation stoichiometry in human cells is approximately 0.02% — meaning that for a typical non-histone acetylation site, fewer than 1 in 5,000 copies of the protein carry the modification at any given time. High-stoichiometry acetylation (>1% occupancy) is predominantly concentrated on nuclear transcriptional regulators and acetyltransferases. This stoichiometry distribution has fundamental implications for data interpretation: a 2-fold fold-change on a site with 0.01% basal occupancy may not be biologically significant, while the same fold-change at a site with 20% occupancy certainly is. Our acetylomics workflows include stoichiometry calculations alongside relative quantification — providing the occupancy context necessary to interpret fold-change data correctly.

N-Terminal Acetylation

N-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes — occurring co-translationally on 50–80% of human proteins via N-terminal acetyltransferases (NatA–NatF). NTA affects protein stability (N-end rule pathway), membrane targeting, and protein complex assembly. Unlike Lys acetylation, NTA is largely irreversible and constitutive. Our acetylomics workflow identifies and quantifies N-terminal acetylation separately from Lys acetylation using specific database search parameters for α-amino group acetylation, enabling comprehensive characterization of the NTA proteome alongside the Lys acetylome.

Acetylation & Drug Discovery

HDAC inhibitors (vorinostat/SAHA, romidepsin, entinostat, panobinostat) are approved cancer therapeutics with broad acetylation effects that extend far beyond histone hyperacetylation. KAT (HAT) inhibitors targeting p300/CBP (A-485, CCS1477) and PCAF/GCN5 are in clinical and preclinical development. Sirtuin modulators (SRT2104, nicotinamide) affect NAD⁺-dependent deacylation. For any of these programs, quantitative acetylomics is the definitive analytical tool: identifying all acetylation sites regulated by the drug, establishing dose-response relationships at the site level, detecting off-target deacetylase effects, and characterizing the temporal dynamics of acetylation changes that precede transcriptional reprogramming. Our HDAC/HAT Activity Assay service provides complementary enzymatic activity measurement alongside acetylomics profiling.

Acetylated Peptide Enrichment

Standalone pan-Ac-Lys antibody immunoaffinity enrichment of acetylated peptides from tryptic digests — provided as a sample preparation service for research groups that perform their own LC-MS/MS analysis. Enrichment is performed using validated anti-acetyllysine monoclonal antibody beads at 4 °C under standardized IAP buffer conditions. Enrichment efficiency is monitored per batch using spiked synthetic acetylated peptide standards. Post-enrichment samples are delivered as dried peptides on C18 StageTips, ready for LC-MS/MS injection.

Acetylation Site Identification

Qualitative identification of acetylation sites — determining which lysine residues and N-termini carry acetylation in your sample without quantitative comparison between conditions. Suitable for initial characterization of the acetylome of an unstudied protein, organism, or cell type; for confirming acetylation status of a target protein; or for generating a comprehensive site list prior to targeted quantitative follow-up. Site localization probability (ptmRS ≥0.75) is reported for every identified site.

Quantitative Acetylomics Analysis

Site-level relative quantification of acetylation changes between conditions — applying SILAC, TMT, DIA/SWATH, or label-free strategies to compare acetylation abundance across treatment groups, time points, or disease states. Delivers log2 fold-change, p-value, and adjusted p-value per site, alongside stoichiometry estimates where protein abundance data from pre-enrichment aliquots enables normalization. See the PTM Quantitative Analysis hub for quantification strategy selection guidance.

Histone Acetylation Analysis

Dedicated histone modification profiling workflow using acid extraction, propionylation derivatization (before and after trypsin digestion to improve histone peptide retention and detection), and short-gradient LC-MS/MS optimized for histone peptides. Covers all H3, H4, H2A, and H2B acetylation marks simultaneously — including K9ac, K14ac, K18ac, K27ac, K36ac (H3), K5ac, K8ac, K12ac, K16ac (H4) — with quantitative stoichiometry measurement per mark across conditions. Co-occurring combinatorial modifications (e.g., H3K9ac + H3K14ac on the same peptide) are also characterized. See dedicated Histone PTM Analysis service →

HDAC / HAT Inhibitor Response Acetylomics

Comprehensive acetylomics characterization of cellular response to HDAC or HAT inhibitor treatment — designed for drug MoA studies, target engagement confirmation, and off-target acetylation effect profiling. Uses SILAC or TMT quantification to compare treated vs. DMSO-control acetylomes, with protein normalization to distinguish acetylation-intrinsic changes from protein abundance effects. Time-course and dose-response designs are supported. Integrates with HDAC/HAT Activity Assays and phosphoproteomics from matched samples for combined epigenetic-signaling characterization.

Acetylation Stoichiometry Measurement

Quantification of the fractional occupancy of specific acetylation sites — the percentage of the total protein population carrying the modification at each site. Stoichiometry measurement requires parallel detection of both the acetylated modified peptide and the unmodified counterpart in the same sample, with normalization for protein abundance. Identifies which acetylation events are high-occupancy regulatory switches (>5% stoichiometry) versus low-occupancy background events, providing functional context for fold-change data from differential acetylomics. Applicable to HDAC inhibitor titration studies, cell-cycle stage comparisons, and nutrient perturbation experiments.

Acylation Panel — Acetyl + Emerging Acyl Marks

Extended profiling combining acetylation with other short-chain acyl modifications from the same digest — crotonylation, succinylation, lactylation, propionylation, butyrylation, malonylation, 2-hydroxyisobutyrylation — in a coordinated multi-enrichment workflow. Enables crosstalk analysis between modification types at shared lysine sites. See Acylation Quantitative Proteomics Service for the full multi-acyl panel, or individual pages for Crotonylation, Lactylation, and Succinylation services.

Acetylomics Data Analysis

Bioinformatics-only service for research groups with existing acetylomics raw data requiring expert analysis — re-processing of raw files with current software, site localization scoring with ptmRS, quantitative normalization, statistical testing, stoichiometry calculation, GO/KEGG pathway enrichment, and HDAC/KAT substrate network annotation. Delivered with publication-ready figures and methods text. See Acetylomics Data Analysis Service →

Enrichment Chemistry

Pan-Ac-Lys antibody immunoaffinity enrichment is the primary method for global acetylomics. We use validated pan-acetyllysine monoclonal antibody-bead conjugates (Cell Signaling Technology, cat. #13416 or equivalent validated batch). Tryptic digests are incubated with antibody beads at 4 °C in IAP buffer for 2 h with end-over-end mixing. Three IAP washes and two PBS washes remove non-specifically bound peptides. Enriched acetylated peptides are eluted with 0.15% TFA, desalted on C18 StageTips, and dried for LC-MS/MS.

A key technical consideration: trypsin generates peptides with free Lys C-termini that are unmodified — these can be partially recognized by pan-Ac-Lys antibodies as background. To reduce this artifact and simultaneously improve the retention of hydrophilic histone peptides, our histone acetylomics workflow applies propionylation derivatization before and after trypsin digestion: pre-digestion propionylation caps unmodified and monomethylated lysines (distinguishing them from acetyl-Lys by mass); post-digestion propionylation caps peptide N-termini. This strategy is essential for high-quality histone acetylation stoichiometry data.

Mass Spectrometry Acquisition

Acetylated peptides are analyzed by nanoflow UHPLC (75 µm × 50 cm C18, 90 min gradient) coupled to high-resolution mass spectrometry. Platform selection is matched to the experimental design:

Thermo Orbitrap Fusion Lumos — primary platform for acetylomics requiring high MS2 resolution for site localization, EThcD fragmentation for N-terminal acetylation and labile modifications, and SPS-MS3 for TMT multiplexed acetylomics with reduced ratio compression.

Q Exactive HF-X — for DDA-based SILAC and label-free acetylomics with high scan rates for deep acetylome coverage.

Bruker timsTOF Pro (diaPASEF) — for DIA-based large-cohort acetylomics with ion mobility separation reducing co-elution between acetylated and unmodified isobaric peptides, improving quantitative precision at low stoichiometry sites.

Acetylation (+42.011 Da) is specified as a variable modification on Lys and protein N-termini in all database searches; trimethylation (+42.047 Da) is included as a competing variable modification to distinguish the two isobaric marks by Orbitrap mass accuracy.

Data Analysis & Bioinformatics

Database search: MaxQuant (SILAC/LFQ) or Proteome Discoverer (TMT) with acetyl (+42.011 Da) on Lys and protein N-terminus as variable modifications; trimethyl (+42.047 Da) and propionyl (+56.026 Da) for histone workflows; ptmRS for site localization probability scoring (≥0.75 threshold for confident site assignment).

Quantification: SILAC delivers the highest ratio accuracy for two-condition comparisons in cell lines; TMT-MS3 enables 16-plex multi-condition acetylomics; DIA with spectral library or DIA-NN library-free search provides low-missing-value large-cohort quantification.

Stoichiometry: site occupancy is calculated using the ratio of modified to unmodified counterpart peptide intensities, normalized by the corresponding protein abundance ratio from the pre-enrichment total proteome aliquot.

Functional annotation: regulated sites mapped to HAT/HDAC substrate databases, PhosphoSitePlus acetylation annotations, bromodomain reader recognition motifs, KEGG/Reactome metabolic and signaling pathways. Subcellular localization enrichment analysis identifies compartment-specific acetylation regulation.

Step 1 — Lysis & Protein Extraction

Cells are lysed in urea lysis buffer (8 M urea, 50 mM ammonium bicarbonate) supplemented with protease inhibitors, TSA (1 μM), and nicotinamide (5 mM). Protein concentration is measured by BCA. For SILAC projects, heavy and light lysates are mixed at a 1:1 protein mass ratio. For TMT projects, each sample is processed independently to the peptide stage before pooling after labeling. A 5% pre-enrichment aliquot is retained for total proteome quantification to enable stoichiometry and protein normalization calculations.

Step 2 — Reduction, Alkylation & Digestion

Proteins are reduced with DTT (10 mM, 30 min, 37 °C), alkylated with chloroacetamide (25 mM, 30 min, RT, dark), diluted to ≤2 M urea, and digested with sequencing-grade trypsin (1:50 enzyme:protein, overnight, 37 °C). For histone acetylomics, propionylation derivatization is applied before digestion (to cap unmodified and monomethylated Lys) and after digestion (to cap peptide N-termini). Digestion completeness is assessed by LC-UV before enrichment. For TMT projects, isobaric labeling is performed on dried, desalted peptides before pan-Ac-Lys enrichment.

Step 3 — Pan-Ac-Lys Immunoaffinity Enrichment

Lyophilized peptides are reconstituted in IAP buffer and incubated with anti-Ac-Lys antibody-bead conjugate at 4 °C for 2 h with end-over-end rotation. Non-specifically bound peptides are removed by sequential washes (three IAP, two PBS). Acetylated peptides are eluted with 0.15% TFA, desalted on C18 StageTips, and lyophilized for LC-MS/MS. Enrichment efficiency is monitored using a synthetic acetylated peptide standard spiked before the IAP step; we report enrichment specificity (% acetylated peptides in eluate) and recovery as QC metrics.

Step 4 — LC-MS/MS Acquisition

Enriched acetylated peptides are analyzed by nanoflow UHPLC-MS/MS on the platform matched to the quantification strategy (Fusion Lumos for SILAC/TMT-MS3; Q Exactive HF-X for DDA-LFQ; timsTOF Pro for diaPASEF-DIA). Database searches include acetyl (+42.011 Da, Lys and N-term), trimethyl (+42.047 Da, Lys), propionyl (+56.026 Da, for histone workflows), carbamylation, oxidation, and deamidation as variable modifications; 1% FDR at PSM and protein level; ptmRS site localization scoring with ≥0.75 threshold for confident site reporting. Acetylation vs. trimethylation disambiguation is confirmed at Orbitrap mass accuracy for ambiguous sites.

Step 5 — Quantitative Analysis & Stoichiometry

Site-level normalized intensities (LFQ/DIA) or isotope ratios (SILAC) or reporter ion intensities (TMT) are extracted and compared between conditions. Protein abundance ratios from the pre-enrichment proteome aliquot are used to correct acetylation ratios for protein expression changes — delivering modification-intrinsic acetylation fold-changes. Stoichiometry is calculated per site as: (acetylated peptide intensity / (acetylated + unmodified counterpart intensity)) × 100%. Statistical testing: t-test or limma for small sample sizes; Benjamini-Hochberg FDR correction; significance thresholds: |FC| ≥1.5 and padj ≤0.05 as default (adjustable per project).

Step 6 — Bioinformatics & Reporting

Regulated acetylation sites are annotated against PhosphoSitePlus, the Histone Modification Database, and KAT/KDAC substrate databases. GO biological process, molecular function, and KEGG pathway enrichment is performed on proteins with significantly regulated acetylation. Subcellular localization enrichment identifies compartment-specific regulation (nuclear chromatin, mitochondria, cytoplasm). HAT/HDAC substrate motif enrichment analysis identifies which writer/eraser enzyme families are most affected. Deliverables: raw data files, site identification and quantification tables, stoichiometry matrix, differential analysis results, bioinformatics figures (volcano, heatmap, GO/KEGG bubble plots, subcellular enrichment, substrate network), and comprehensive project report with publication-ready methods text.

Background & Scientific Question

Thousands of acetylation sites had been identified in human cells by the time of this study, but a fundamental parameter remained poorly characterized: what fraction of each protein population is actually acetylated at any given lysine site? This stoichiometry question is biologically critical — a 2-fold fold-change in a site with 20% basal occupancy has different mechanistic significance than the same fold-change in a site with 0.005% basal occupancy. Without stoichiometry data, it is impossible to determine which acetylation events are high-occupancy regulatory switches versus which represent nonenzymatic background acetylation unlikely to contribute to protein regulation. Hansen, Gupta, Choudhary, and Weinert at the University of Copenhagen set out to measure acetylation stoichiometry at proteome scale in human cells — providing the first comprehensive, quantitatively validated stoichiometry map of the human acetylome.

Methods

To measure stoichiometry, both the acetylated and the unmodified counterpart peptide for each site must be quantified in the same experiment. The study used a spike-in SILAC strategy: chemically acetylated heavy-labeled reference peptides were spiked into HeLa cell lysates at known concentrations, enabling absolute quantification of both acetylated and unmodified endogenous peptides via their ratios to the heavy reference. Pan-Ac-Lys antibody enrichment was combined with unenriched total proteome analysis from the same samples to provide the protein-level normalization needed to convert peptide ratios into percentage occupancy values. HDAC inhibitors (trichostatin A for class I/II HDACs, tubacin for HDAC6, nicotinamide for Sirtuins) were applied to map which sites are regulated by specific deacetylase classes. CBP/p300 HAT inhibitors and chemical genetics approaches identified KAT-regulated sites with elevated stoichiometry.

Results Overview

The study delivered validated stoichiometry measurements for 6,829 acetylation sites on 2,535 proteins — the most comprehensive quantitative acetylome resource available at the time of publication. The key findings:

Stoichiometry is extremely low for most sites: the median stoichiometry across all 6,829 sites is ~0.02% — meaning that most acetylation events in the cell occur on far less than 1% of the protein population. This low-stoichiometry bulk is dominated by nonenzymatic acetylation from acetyl-CoA that is unlikely to serve regulatory functions.

High-stoichiometry acetylation (>1%) is concentrated on nuclear transcriptional regulators: histones, acetyltransferases, and transcription factors that depend on acetylation for function have dramatically higher occupancy than cytoplasmic or mitochondrial proteins. This bimodal distribution explains why acetylation-dependent regulation is most firmly established for nuclear proteins.

CBP/p300 preferentially targets high-stoichiometry sites: KAT3A/B (p300/CBP) substrate sites have significantly higher median stoichiometry than sites regulated by other KATs — consistent with its role as a broad epigenetic "writer" maintaining high-occupancy chromatin acetylation marks. HDAC inhibition raises histone acetylation stoichiometry from ~20–30% to >80% within hours, while most non-histone sites increase by only 1.5–2-fold — revealing the steep dose-response hierarchy between chromatin and non-chromatin acetylation.

Relevance to Our Service

This study defines the analytical framework that our acetylomics stoichiometry service is built around. The finding that most acetylation occurs below 0.05% occupancy means that fold-change data alone — without stoichiometry — cannot distinguish regulatory acetylation from nonenzymatic background: a 3-fold increase from 0.005% to 0.015% occupancy is analytically real but functionally unlikely to be regulatory; a 3-fold increase from 10% to 30% occupancy is both measurable and almost certainly mechanistically significant.

Our Acetylation Stoichiometry Measurement service implements the same principle: parallel measurement of acetylated and unmodified counterpart peptides with protein-level normalization, delivering percentage occupancy values per site alongside relative fold-change data. For HDAC inhibitor characterization studies, our quantitative acetylomics workflow captures both the chromatin-level hyperacetylation response and the non-histone site regulation — providing the complete acetylome MoA readout that the data in this study established as the field standard.