Analysis of Multi-Pathway Phosphoproteomics Using Orbitrap Fusion Lumos

Phosphorylation, an important post-translational modification, is widely involved in various biological processes such as cell signaling, metabolic regulation, and cell cycle control. It introduces phosphate groups to tyrosine, serine, and threonine residues through enzymatic reactions, regulating protein activity, stability, and interactions. In recent years, with the rapid development of high-throughput mass spectrometry technologies, especially the application of advanced instruments like Orbitrap Fusion Lumos, research in phosphorylation proteomics has become more efficient and precise.

 

Technical Features of Orbitrap Fusion Lumos

The Orbitrap Fusion Lumos mass spectrometer has multiple advantages that make it excel in phosphorylation proteomics analysis. Firstly, its high resolution and sensitivity enable the analysis of trace samples, which is crucial for detecting phosphorylation modifications. Secondly, the Fusion Lumos combines three different fragmentation methods, including HCD (higher-energy collisional dissociation), CID (collision-induced dissociation), and ETD (electron transfer dissociation), allowing for optimal dissociation strategies tailored to different types of peptides, thereby improving the detection rate of phosphorylation sites.

 

Workflow of Multi-Pathway Phosphorylation Proteomics Analysis

1. Sample Preparation

Before conducting phosphorylation proteomics analysis, sample preparation is a critical step. First, total proteins need to be extracted from cells or tissues. Then, enzymatic digestion (typically using trypsin) is performed to cut proteins into peptides. To enhance the recovery of phosphorylated peptides, selective enrichment strategies are often employed, such as using TiO2 or iron-sulfur catalysts for selective capture of phosphopeptides.

 

2. Mass Spectrometry Analysis

In mass spectrometry analysis using Orbitrap Fusion Lumos, the sample is first injected into a nanoscale liquid chromatography system (nLC). After separation of peptides in the chromatographic column, they enter the mass spectrometer and are analyzed based on their mass-to-charge ratio (m/z). The high resolution of the Fusion Lumos enables the differentiation of phosphorylated and non-phosphorylated peptides in complex samples.

 

3. Data Acquisition and Analysis

During the data acquisition phase, a data-dependent acquisition (DDA) mode is employed, which allows real-time monitoring of ion signals and selects the most intense peptides for further fragmentation and analysis. This maximizes the detection sensitivity for phosphorylated peptides. Simultaneously, software such as PEAKS or MaxQuant is used for data analysis, enabling qualitative and quantitative analysis of phosphorylation sites.

 

4. Phosphorylation Site Identification

Through mass spectrometry data, phosphorylation sites can be accurately identified. Generally, the m/z values of peptides, the fragmentation patterns of ions, and their relative abundances provide crucial information to help researchers determine the phosphorylation position and the corresponding peptide sequence. By combining bioinformatics tools, it is possible to further elucidate the role of phosphorylation in cellular signaling pathways.

 

5. Result Validation

Finally, for the identified phosphorylation sites, further validation can be conducted. Common methods include Western blotting and enzyme-linked immunosorbent assay (ELISA), which can provide reliable support for the mass spectrometry results.

 

Using Orbitrap Fusion Lumos for multi-pathway phosphorylation proteomics analysis not only enhances the sensitivity and accuracy of phosphorylated peptide detection but also provides a powerful tool for understanding the complex signaling mechanisms within cells.