How to Choose the Right Enrichment Strategy for Acylated Peptides?
- Low modification abundance, with signals easily masked by unmodified peptides
- Co-existence of multiple, structurally similar acylation types that are difficult to discriminate
- Altered peptide physicochemical properties leading to reduced detectability
- High specificity toward defined acylation types
- Suitable for high-throughput proteomics studies
- Applicable to diverse acylation types
- Amenable to multi-step or tandem enrichment workflows
- Strict control of reaction conditions is required to avoid side reactions
- Workflows are more complex and susceptible to procedural variability
- Highly targeted to enhance coverage and recovery of histone modifications
- Ideal for epigenetic research applications
- Restricted to nuclear extracts from cells or tissues
- Not applicable to non-histone protein studies
- Well-validated antibody libraries covering diverse acylation types
- High-resolution MS instruments (e.g., Orbitrap Eclipse, Exploris 480) for deep proteome profiling
- Standardized analysis pipelines enabling multidimensional PTM interrogation
- Dedicated scientific teams offering tailored project consultation and data interpretation
Protein acylation, an essential post-translational modification (PTM), plays crucial roles in cellular signal transduction, gene regulation, metabolic control, and other biological processes. Common acylation types, including acetylation, propionylation, and butyrylation, occur on both histone and non-histone proteins and are implicated in a wide range of physiological and pathological events such as tumorigenesis, inflammation, and metabolic disorders. However, acyl modifications often exist at low stoichiometry, making direct mass spectrometry (MS) detection challenging with respect to sensitivity and proteome coverage. Therefore, selecting an effective enrichment strategy for acylated peptides prior to MS analysis is essential to ensure data depth and reliability.
Importance of Enriching Acylated Peptides
The analysis of acylation faces several intrinsic challenges:
Appropriate enrichment can significantly increase the identification of acylated peptides while improving quantification accuracy and reproducibility.
Mainstream Enrichment Strategies for Acylation
Current enrichment approaches for acylated peptides exhibit distinct advantages and are suitable for different research scenarios:
1. Antibody-Based Enrichment
(1) Target scope: Lysine acylations such as acetylation, propionylation, and butyrylation
(2) Principle: Modification-specific antibodies (e.g., anti-acetyl-lysine) selectively bind acylated lysines and enable immunoprecipitation-based enrichment.
(3) Advantages
(4) Limitations
Considerable variability in antibody quality and limited batch-to-batch consistency
Ineffective for rare or uncharacterized acylation types
2. Chemical Derivatization-Based Enrichment
(1) Target scope: Broad-range acylations and low-abundance modifications
(2) Principle: Chemical reactivity of acyl groups is exploited for selective tagging or capture, such as hydrazide-based solid-phase coupling of acylated peptides.
(3) Advantages
(4) Limitations
3. Histone Extraction and Enrichment
(1) Target scope: Histone acylations (e.g., H3K27ac, H4K16ac)
(2) Principle: Enrichment begins with selective extraction of histones via acid or salt extraction, followed by antibody-based or chromatographic enrichment.
(3) Advantages
(4) Limitations
Criteria for Selecting an Enrichment Strategy
1. Modification Characteristics and Study Goals
(1) Well-characterized modifications (e.g., acetylation): antibody-based enrichment preferred
(2) Novel or undefined acylations: chemical derivatization recommended to broaden detection scope
2. Sample Source and Complexity
(1) Histone-focused studies: histone extraction combined with targeted enrichment
(2) Heterogeneous or non-histone samples: chemical or multi-step enrichment strategies recommended
3. Reagent Availability and Instrument Capability
(1) Antibody workflows require validated antibody-bead systems
(2) Chemical derivatization benefits from high-resolution MS platforms
4. Budget and Throughput Requirements
(1) High-throughput proteomics: robust, reproducible antibody-based workflows
(2) Exploratory studies: dual-strategy or comparative approaches for enhanced coverage
MtoZ Biolabs: Integrated Solutions for PTM-focused Proteomics
The choice of enrichment strategy directly impacts MS performance in terms of depth, accuracy, and reproducibility. Leveraging extensive proteomics expertise, MtoZ Biolabs has established standardized platforms encompassing antibody-based enrichment, chemical derivatization, and histone-specific workflows to support customized experimental designs.
Our capabilities include:
To decode PTM regulatory mechanisms, method selection of acylated peptides should integrate modification type, sample nature, and research objectives. Antibody-based enrichment remains the preferred option for common acylations, whereas chemical derivatization provides enhanced flexibility for emerging modifications and exploratory studies. Through MtoZ Biolabs’ comprehensive platform, researchers can acquire reliable and high-performance acyl-proteomics solutions.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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