Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
April 4th, 2018
Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.
The overall goal of this novel workflow is to determine the occupancy or stoichiometry of protein post-translational modifications at specific acetylation sites.This novel mass spectrometric workflow provides a higher level of precision of quantification.This method can help answer key questions about reversible protein regulation by post translational modification such as lysine acetylation or succinylation sites are most highly modified and may alter protein function.The main advantage of this technique is that site level stoichiometry of lysine acetylation and succinylation and succinylation are accurately determined and the method can determine stoichiometry from peptides with multiple lysine residues.This technique is highly relevant because it reveals acetylation and succinylation stoichiometry or proportion modified versus unmodified instead of measuring a dimensionless relative abundance change.I first had the idea for this method when I realized that using data independent acquisitions and MS2 based quantification for stoichiometry analysis would greatly improve measurement accuracy.Prepare three replicates of 100
Overview
This study presents a novel mass spectrometric workflow for quantifying protein post-translational modifications, specifically acetylation and succinylation. The method enhances measurement accuracy by determining site-specific occupancy or stoichiometry of these modifications.
Key Study Components
Area of Science
- Biochemistry
- Mass Spectrometry
- Proteomics
Background
- Post-translational modifications play a crucial role in protein function.
- Understanding acetylation and succinylation is vital for studying reversible protein regulation.
- Traditional methods may lack precision in quantifying these modifications.
- This study aims to improve the accuracy of stoichiometry measurements.
Purpose of Study
- To develop a workflow for accurate quantification of protein acetylation and succinylation.
- To identify which lysine sites are most modified and their impact on protein function.
- To provide a method that can analyze peptides with multiple lysine residues.
Methods Used
- Ratiometric analysis of endogenous modifications.
- Quantitative chemical acylation using stable isotope-labeled anhydrides.
- Data-independent acquisition mass spectrometry.
- Preparation of multiple replicates for accuracy.
Main Results
- The method allows for precise determination of site-level stoichiometry.
- It reveals the proportion of modified versus unmodified lysine residues.
- Higher measurement accuracy compared to traditional methods.
- Identifies key acetylation and succinylation sites affecting protein function.
Conclusions
- This workflow significantly enhances the quantification of protein modifications.
- It provides insights into the regulation of protein function through acetylation and succinylation.
- The technique is applicable to a wide range of proteomic studies.
Frequently Asked Questions
Executive Industry Relevance
Accurate quantification of site-specific lysine acetylation and succinylation stoichiometry enables precise assessment of post-translational modification occupancy, which is critical for understanding reversible protein regulation in drug target pathways. This method provides unbiased, proteome-wide measurement of modification levels, supporting target validation by distinguishing biologically relevant acylation sites from background noise. By delivering stoichiometric data rather than relative abundance changes, it enhances predictive confidence in mechanistic de-risking during early discovery.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying occupancy of acetylation and succinylation at specific lysine residues.
- Operational Value: Supports biological de-risking through precise measurement of modification stoichiometry, clarifying functional impact on protein activity.
- Predictive Value: Improves target confidence by identifying which modified sites are most likely to alter function, aiding in portfolio triage.
Screening & Assay Development
- Assay Readiness: Prepares validated biological systems for downstream workflows by providing site-level acylation occupancy data.
- Quantitative Output: Uses fragment ion peak areas from DIA-MS to enable accurate, interference-reduced quantification suitable for assay standardization.
- Scalability: Facilitates screening readiness through reproducible, multiplex-capable proteomic analysis of acetylation and succinylation.
Translational & Preclinical Research
- Translational Continuity: Connects discovery-phase modification profiling to preclinical validation by identifying disease-relevant acylation sites.
- Mechanistic De-risking: Focuses on predictive value by determining stoichiometry from peptides with multiple lysine residues, enabling site-specific resolution.
- Risk-Adjusted Advancement: Supports go/no-go decisions by revealing which lysine modifications are functionally significant versus stochastic.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from early target validation through lead identification, providing stoichiometric acetylation and succinylation data that inform mechanistic understanding and compound effects on protein function.
- Discovery Biology: Supports hypothesis testing and pathway clarification by quantifying site-specific modification occupancy in response to perturbations.
- Screening: Enables assay readiness through standardized, quantitative measurement of modification levels across proteomic samples.
- Analytics: Delivers precise stoichiometry measurements via DIA-MS fragment ion quantification, allowing accurate comparison of modification states.
- Translational Research: Links to preclinical continuity by identifying acylation sites with high occupancy that may serve as biomarkers or mechanistic readouts.
- Enterprise Reuse: Functions as a reusable proteomic capability for consistent PTM profiling across projects and target classes.
Operational & Enterprise Impact
- Scientific Value: Provides predictive confidence through accurate site-level stoichiometry of acetylation and succinylation, reducing mechanistic ambiguity in target validation.
- Operational Value: Ensures standardization and reproducibility via DIA-MS and Skyline-based quantification, minimizing technical variability.
- Strategic Value: Improves go/no-go decisions by identifying high-occupancy, functionally relevant modification sites, reducing late-stage biological risk.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on modification-driven functional evidence.
Implementation Considerations
- Requires expertise in mass spectrometry-based proteomics and PTM enrichment techniques.
- Dependence on DIA-capable instrumentation and spectral library generation for accurate fragment ion quantification.
- Necessitates cross-team standardization of sample preparation and chemical acylation protocols for reproducibility.
- Involves adaptation considerations when applying the workflow to diverse model systems or tissue types.
- Involves practical limitations related to peptide detectability and lysine residue accessibility in complex mixtures.
Biopharma FAQs
Chapters in this video
0:04
Title
1:18
Quantitatively Acetylate and/or Succinylate Proteins Using Isotope-labeled Acetic or Succinic Anhydride
3:48
Digest Reacted Protein Samples Using Glu-C Endoproteinase
6:27
Fractionate Peptides Using Offline Basic-pH Reversed Phase HPLC
8:15
Example Data Analysis Tutorial
10:28
Results: Succinylation Stoichiometry Ratio Calculation of Pre-succinylated BSA
11:13
Conclusion
