How to Perform High-Throughput Protein Acylation Analysis?
- Cells or tissues are lysed in buffers containing deacylase inhibitors (e.g., TSA, NAM) to prevent modification loss.
- Proteins are then quantified following SDS- or urea-based lysis and subsequently digested using either FASP or in-solution protocols.
- Antibody enrichment: Employ antibodies specific to acyl modifications (e.g., anti-Kac, anti-Ksucc) for immunoaffinity purification.
- Multiplex enrichment: Combine multiple antibodies in tandem or integrate with HILIC-based pretreatment to improve specificity.
- TMT labeling with enrichment: Enables multiplexed sample analysis, increasing both throughput and efficiency.
- Common instruments include Orbitrap Exploris, Q Exactive HF-X, and TIMS-TOF Pro.
- Data-dependent acquisition (DDA) allows deep proteome coverage, whereas data-independent acquisition (DIA) enhances reproducibility and quantitative completeness.
- Multi-stage fragmentation (MS/MS or MS³) improves site localization accuracy.
- Data processing typically employs search engines such as MaxQuant, Proteome Discoverer, Spectronaut (for DIA), or PEAKS.
- Modification definitions follow the Unimod database.
- Mass shifts must be set accordingly (e.g., +42.0106 Da for Kac).
- Localization confidence is evaluated using algorithms such as ptmRS or Ascore.
- Refine database search parameters to avoid ambiguity
- Employ high-resolution MS/MS to resolve isomeric peptides
- Use sequential or tandem antibody enrichment workflows
- Perform total proteome analysis in parallel
- Normalize acylation levels against protein expression to reveal genuine regulatory events
- Integrate TMT labeling with immuno-enrichment to achieve 16-plex or 18-plex analyses
- Adopt DIA strategies with pooled-sample designs for large-scale screening
Protein acylation represents a major class of post-translational modifications (PTMs) that play crucial roles in regulating cellular metabolism, signal transduction, and chromatin remodeling. Recent advances in proteomics, particularly in high-resolution mass spectrometry, have enabled high-throughput profiling of protein acylation. This capability has driven rapid progress in research areas such as metabolic regulation, epigenetics, and biomarker discovery in cancer.
What Is Protein Acylation?
Protein acylation refers to the covalent attachment of an acyl group (e.g., acetyl, propionyl, butyryl, malonyl, or succinyl) to proteins, typically at lysine residues (K) or the N-terminal amino group. Among these, lysine acetylation (Kac) is the most extensively characterized modification. However, emerging “non-classical” acylations, including propionylation (Kpr), butyrylation (Kbu), succinylation (Ksucc), and malonylation (Kmal), have attracted growing scientific interest. These modifications are closely linked to the metabolic state of the cell, as their donors (e.g., acetyl-CoA and succinyl-CoA) are direct intermediates of metabolic pathways. Therefore, acylation is regarded as a critical molecular bridge connecting cellular metabolism and epigenetic regulation.
Overview of High-Throughput Protein Acylation Analysis
High-throughput acylation analysis primarily relies on mass spectrometry, combined with antibody-based enrichment, specific proteolytic digestion, and multidimensional separation strategies. The general workflow consists of the following steps:
1. Sample Preparation and Protein Extraction
2. Enrichment of Acyl-Modified Peptides
Given that acylated peptides constitute less than 0.1% of total peptides, highly selective enrichment methods are essential for sensitive detection:
3. High-Resolution Mass Spectrometric Analysis
4. Data Analysis and Site Identification
Challenges and Solutions in Parallel Protein Acylation Analysis
1. Multiple Modification Types and Isotopic Interference
The small mass differences among acylations (e.g., Kbu +56.026 Da vs. Ksucc +100.017 Da) can cause quantification overlap.
Solutions:
2. Confounding Effects of Protein Abundance
Changes in acylation levels do not necessarily imply functional relevance, as they may reflect changes in total protein abundance.
Solutions:
3. Limited Sample Throughput
Conventional DDA-based workflows typically process fewer than ten samples per batch.
Solutions:
Protein acylation serves as a crucial link between metabolic signaling and protein function, and its systematic investigation is essential for understanding complex biological processes. By integrating high-resolution mass spectrometry, specific antibody-based enrichment strategies, and multiplex quantitative approaches, researchers can now efficiently perform parallel analyses of diverse acylation modifications. Leveraging its advanced mass spectrometry platforms and well-established acylomics workflows, MtoZ Biolabs provides comprehensive analytical solutions that empower scientists to achieve breakthrough discoveries in metabolic epigenetics, disease mechanism elucidation, and target identification. Researchers interested in exploring related topics are welcome to contact us for customized project proposals or detailed technical documentation.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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