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
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
