Analysis of Methylated Proteins Using Orbitrap Fusion Lumos

Methylation is a critical post-translational modification (PTM) of proteins that plays an essential role in regulating gene expression, cell signaling, and protein function. Protein methylation primarily occurs on lysine and arginine residues, and its dynamic changes are closely associated with the development of various diseases, such as cancer and neurodegenerative disorders. Therefore, comprehensive analysis of methylated proteins is of significant research importance. The Orbitrap Fusion Lumos mass spectrometer, as a high-resolution and high-sensitivity mass spectrometry tool, has been widely applied in methylated proteomics studies in recent years.

 

Basic Principles and Features of Orbitrap Fusion Lumos

The Orbitrap Fusion Lumos mass spectrometer, developed by Thermo Fisher Scientific, combines multiple mass spectrometry technologies, including Orbitrap and linear ion trap. Its characteristics of high resolution, high sensitivity, wide dynamic range, and fast scanning speed enable precise detection and quantification of protein methylation modifications.

 

1. Multi-Stage Mass Spectrometry (MSn) Capability

Orbitrap Fusion Lumos can perform multi-stage mass spectrometry analysis, providing detailed molecular structural information through multiple rounds of fragmentation and detection. This capability is particularly crucial for complex methylation modification studies, as the identification of different methylation sites requires high-resolution fragment ion information.

 

2. High Resolution and Mass Accuracy

The high resolution and mass accuracy of Orbitrap Fusion Lumos effectively distinguish structurally similar modifications, such as between monomethylation and dimethylation. This resolving power significantly improves the accuracy of methylation modification identification.

 

3. High Sensitivity

The high sensitivity of this mass spectrometer allows for the detection of low-abundance methylated peptides, revealing modification sites that are lowly expressed but functionally significant in cells.

 

Workflow of Analyzing Methylated Proteins Using Orbitrap Fusion Lumos

The analysis of methylated proteins using Orbitrap Fusion Lumos generally involves three main steps: sample preparation, mass spectrometry detection, and data analysis.

 

1. Sample Preparation

Initially, proteins are digested into peptides using enzymes, and methylation-specific enrichment methods, such as immunoprecipitation or antibody capture, are used to enrich target methylated peptides. Each step in the sample preparation process can affect the final mass spectrometry results, so strict control is required.

 

2. Mass Spectrometry Detection

During the mass spectrometry detection phase, Orbitrap Fusion Lumos, with its multi-stage mass spectrometry capability and high resolution, can accurately identify methylated peptides. Mass spectrometry data collection typically employs Data-Dependent Acquisition (DDA) or Data-Independent Acquisition (DIA) modes; the former provides detailed fragment ion information, while the latter helps comprehensively cover peptides in the sample.

 

3. Data Analysis

Data analysis of mass spectrometry is a crucial step in methylated protein research. Specialized software, such as Proteome Discoverer or MaxQuant, is used to process the collected mass spectrometry data, including peptide identification, modification site localization, and quantitative analysis. These data analysis tools, combined with database searches and modification feature filtering, can accurately identify methylation modifications and assess their changes under different biological conditions.

 

Applications of Orbitrap Fusion Lumos in Methylation Research

In recent years, the use of Orbitrap Fusion Lumos to analyze methylated proteins has achieved remarkable results in various research fields. For example, in cancer research, abnormal changes in methylation modifications have been found to be closely related to the occurrence, development, and metastasis of cancer. Through Orbitrap Fusion Lumos mass spectrometry analysis, researchers can detail the patterns of methylated proteins in cancer cells, revealing potential pathogenic mechanisms.

 

The Orbitrap Fusion Lumos mass spectrometer, with its high resolution, multi-stage mass spectrometry capability, and high sensitivity, is a powerful tool for analyzing methylated proteins. Through precise identification and quantification of methylation modifications, researchers can gain in-depth insights into the roles of protein methylation in various biological processes, providing essential scientific evidence for disease mechanism research and the development of novel therapeutic strategies.