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Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

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Published on: September 14, 2014

Method for Quantifying Oxidized Methionines and Application to HIV-1 Env.

Joshua T Shipman¹, Eden P Go¹, Heather Desaire²

¹Department of Chemistry, University of Kansas, Lawrence, KS, 66045, USA.

Journal of the American Society for Mass Spectrometry

|July 11, 2018

Summary

This study presents a new method to accurately quantify methionine oxidation in proteins, even those with multiple methionine residues. The approach is faster and requires less protein, successfully analyzing HIV-1 Env oxidation.

Keywords:

Env HIV Mass spectrometry Methionine oxidation PTMs post-translational modification Quantitation Stable isotope labeling

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Area of Science:

Biochemistry
Analytical Chemistry
Protein Science

Background:

Recombinant proteins are prone to methionine oxidation during various stages.
Existing methods may overestimate oxidation due to sample preparation artifacts.
Previous methods struggled with peptides containing multiple methionine residues.

Purpose of the Study:

To develop and validate a novel method for quantifying pre-sample preparation methionine oxidation.
To enable accurate analysis of proteins with multiple methionine residues.
To apply the method to a critical HIV vaccine candidate protein.

Main Methods:

Utilized theoretical isotope distributions and experimental spectra.
Employed an 18O-labeled hydrogen peroxide reagent (as in prior work, but adapted).
Validated the method using model proteins and recombinant HIV-1 Env.

Main Results:

Developed a rapid method requiring less protein for analysis.
Successfully quantified methionine oxidation in peptides with multiple methionine residues.
Determined the oxidation state of recombinant HIV-1 Env, which was not possible previously.

Conclusions:

The new method accurately quantifies pre-existing methionine oxidation.
This approach overcomes limitations of previous methods for complex peptides.
The validated method is crucial for analyzing the oxidation state of HIV vaccine candidates like HIV-1 Env.