Detection of Protein Oxidative Modifications Based on Obitrap Fusion Lumos Mass Spectrometry

Protein oxidative modifications (OM) refer to the chemical modifications of protein amino acid residues induced by reactive oxygen species (ROS) and other reactive molecules under oxidative stress conditions. These modifications play significant roles in various biological processes, such as cell signaling, inflammation, and aging. Detecting protein oxidative modifications is crucial for understanding their functions in pathological and physiological processes. In recent years, mass spectrometry (MS), particularly high-resolution instruments like the Obitrap Fusion Lumos, has become a powerful tool for studying protein oxidative modifications.

 

Advantages of Obitrap Fusion Lumos Mass Spectrometry

The Obitrap Fusion Lumos is a high-sensitivity, high-resolution mass spectrometer that combines the advantages of linear ion trap, quadrupole, and Obitrap detectors. It provides precise mass-to-charge ratios (m/z) and a broad dynamic range, making it particularly suitable for the analysis of oxidative modifications in proteomics research. Key features of this platform include:

 

1. High Resolution

With its Obitrap detector, the mass resolution can reach several hundred thousand, enabling clear distinction between oxidatively modified peptides and unmodified ones.

 

2. High Sensitivity

It can detect low-abundance oxidatively modified proteins, making it ideal for studying modifications in samples with low concentrations.

 

3. Multi-Mode Scanning

The combination of collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) provides comprehensive identification of oxidative modification sites.

 

Workflow for Detecting Protein Oxidative Modifications Using Obitrap Fusion Lumos

The detection of protein oxidative modifications typically involves three main steps: sample preparation, mass spectrometry detection, and data analysis.

 

1. Sample Preparation

Sample preparation is critical for mass spectrometry detection. First, target proteins are extracted from cells or tissues using standard protein extraction methods. Next, the proteins are enzymatically digested into peptides using proteases like trypsin, followed by the removal of interfering substances using hydrophilic reagents such as formic acid. To enhance the detection sensitivity of modification sites, enrichment techniques such as immunoprecipitation (IP) or affinity capture are often employed to selectively isolate oxidatively modified peptides.

 

2. Mass Spectrometry Detection

On the Obitrap Fusion Lumos platform, mass spectrometry analysis is typically performed using either data-dependent acquisition (DDA) or data-independent acquisition (DIA) modes. Through MS detection, peptides containing oxidative modification sites are detected based on their characteristic m/z and fragment ion spectra. The combination of HCD and CID enhances the precise identification of modification positions. The identification of oxidative modification forms, such as hydroxylation, nitration, and disulfide bond formation, relies on high-resolution mass spectra.

 

3. Data Analysis

Data analysis is the most complex part of the process. Using specialized mass spectrometry data analysis software (such as Proteome Discoverer), researchers can match the obtained mass spectrometry data with protein sequences in databases to identify specific oxidative modification sites and types. Particularly, with the integrated modification filters and peptide identification algorithms, it is possible to rapidly identify specific oxidatively modified peptides in complex samples. Additionally, quantitative techniques such as SILAC or TMT labeling can be applied to quantify the abundance changes of oxidative modifications.

 

Applications of Obitrap Fusion Lumos in Protein Oxidative Modification Detection

Obitrap Fusion Lumos mass spectrometry has wide applications in oxidative modification research. It can not only detect common modifications like hydroxylation and nitration but also identify rare types of oxidative modifications. Furthermore, the technology is used to study the dynamic changes of post-translational modifications under oxidative stress conditions, helping to uncover the cellular response mechanisms to environmental stress.

 

The detection of protein oxidative modifications using Obitrap Fusion Lumos mass spectrometry is an essential technique in proteomics research today. Its high resolution and sensitivity allow researchers to perform accurate, quantitative analyses of protein oxidative modifications, providing valuable insights into functional changes of proteins under different pathological and physiological conditions.