How to Enrich Low-Abundance Histone Succinylated Peptides?
In recent years, advances in epigenetics and protein post-translational modification research have made histone modifications an important focus in life science research. In addition to classical acetylation, methylation, and phosphorylation, succinylation has attracted increasing attention as an emerging acylation modification because of its important roles in chromatin architecture and gene expression regulation. However, compared with more abundant modifications, succinylation is typically characterized by low abundance, rapid dynamic changes, and substantial analytical challenges. In histone samples in particular, succinylation sites account for only a very small proportion of all lysine modification sites. Therefore, efficient enrichment of low-abundance histone succinylated peptides is a critical step for obtaining high-quality mass spectrometry data. This article systematically introduces the research background of histone succinylation, the challenges associated with low-abundance detection, and current mainstream enrichment strategies, providing practical guidance for experimental design.
Overview of Histone Succinylation
Histone succinylation is an acylation modification that occurs on lysine residues, with succinyl-CoA from cellular metabolism serving as the acyl donor. Compared with acetylation, succinylation converts the lysine side chain from a positively charged state to a negatively charged state. This charge reversal can markedly affect histone-DNA binding, nucleosome stability, and chromatin accessibility, thereby regulating transcription factor recruitment and gene expression. Studies have shown that succinylation plays important roles in tumorigenesis, cellular metabolic reprogramming, inflammatory responses, neurodegenerative diseases, and stem cell fate regulation. Therefore, precise identification and quantitative analysis of histone succinylation sites are of considerable value for life science research.
Challenges in Detecting Low-Abundance Histone Succinylated Peptides
1. Extremely Low Modification Abundance
In total cellular proteins, unmodified peptides constitute the vast majority of the peptide population. Common modifications such as acetylation and phosphorylation are relatively more abundant, whereas succinylated peptides usually account for less than 0.1%. During direct mass spectrometry analysis, large numbers of high-abundance peptides can suppress the ionization efficiency of low-abundance succinylated peptides.
2. Complex Histone Structure
The N-terminal regions of histones are rich in lysine and arginine residues. After tryptic digestion, histones can generate a large number of short peptides. These peptides often have limited sequence length and complex charge states, resulting in weak mass spectrometric responses and further increasing the difficulty of detection.
3. Pronounced Biological Dynamics
Succinylation levels are influenced by metabolic status, oxidative stress, hypoxic conditions, and changes in enzymatic activity. This modification also exhibits pronounced spatiotemporal specificity. As a result, many functionally important sites may be present at extremely low abundance in biological samples.
Therefore, without enrichment, detection depth is limited, site coverage is low, and quantitative accuracy may decrease. Studies have shown that unenriched whole-proteome LC-MS/MS analysis usually identifies only dozens of succinylation sites, whereas specific enrichment can enable the detection of hundreds to thousands of sites, substantially improving detection sensitivity and quantitative reproducibility.
Enrichment Strategies for Low-Abundance Histone Succinylated Peptides
1. Immunoaffinity Enrichment Using Anti-Succinylation Antibodies
At present, immunoaffinity enrichment using anti-Ksucc antibodies is the most widely used strategy. This method relies on antibodies that specifically recognize lysine-succinylated peptides, followed by capture with agarose beads or magnetic beads to enrich the target peptides. The general workflow includes protein extraction, histone isolation, enzymatic digestion, incubation with anti-Ksucc antibodies, elution, and LC-MS/MS analysis. The major advantage of this method is its high enrichment specificity, which enables effective removal of unmodified peptides and non-target modified peptides, thereby substantially improving detection sensitivity. It is also compatible with multiple mass spectrometry strategies, including DDA, DIA, PRM, and TMT-based quantitative workflows. However, antibody quality directly affects experimental performance. Non-specific binding and batch-to-batch variation may compromise data reliability; therefore, rigorously validated high-affinity antibodies should be selected.
2. Histone Pre-Enrichment
Before succinylation enrichment, purification of histones can significantly reduce interference from background proteins. Acid extraction is a commonly used method for histone isolation. In this approach, histones are extracted after cell lysis by adding sulfuric acid solution and are then recovered by trichloroacetic acid precipitation. This method can effectively remove cytoplasmic proteins, membrane proteins, and other high-abundance background proteins, thereby improving the efficiency of subsequent enrichment. It is particularly suitable for histone modification omics and nuclear protein modification analysis.
3. High-pH Reversed-Phase Prefractionation
For complex samples, low-abundance sites may still be missed even after a single round of antibody enrichment. High-pH reversed-phase liquid chromatography can be used to fractionate enzymatically digested peptides into multiple fractions, followed by Ksucc enrichment and LC-MS/MS analysis of each fraction. This approach reduces sample complexity, increases MS/MS sampling opportunities, and improves the probability of identifying low-abundance peptides. Studies have shown that this strategy can increase the number of identified succinylation sites by more than 2- to 5-fold.
4. Sequential Enrichment Strategy
For extremely low-abundance succinylation sites, sequential enrichment is an effective strategy. Histones can first be purified by SDS-PAGE, followed by enzymatic digestion, Ksucc antibody enrichment, and subsequent mass spectrometry analysis. For clinical tissue samples, rare cell samples, or limited-input samples, this strategy can minimize background interference and improve detection sensitivity.
5. DIA Combined with Deep Enrichment
With the development of Data Independent Acquisition (DIA), its high data completeness, good quantitative reproducibility, and low missing-value rate make it particularly suitable for detecting low-abundance succinylated peptides. By first performing Ksucc antibody enrichment, followed by DIA data acquisition and spectral library-based analysis, researchers can further improve site coverage and quantitative accuracy, thereby generating reliable data for biological functional interpretation.
As an emerging epigenetic modification, histone succinylation plays important roles in gene expression regulation, cellular metabolic reprogramming, and disease initiation and progression. Drawing on a mature post-translational modification omics platform and extensive project experience, MtoZ Biolabs provides one-stop services covering sample preparation, succinylated peptide enrichment, mass spectrometry detection, and data analysis, supporting in-depth investigation of the biological functions of succinylation.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
