Phosphorylation Site Mass Spectrometry

Protein phosphorylation is a common post-translational modification, which can regulate many biological processes in cells, including cell proliferation, cell apoptosis, and cell differentiation. In eukaryotes, phosphorylation mainly occurs at serine, threonine, and tyrosine residues. In bacteria, proteins are mainly phosphorylated through aspartic acid, glutamic acid, and histidine residues. Most phosphorylated proteins contain more than one phosphorylation site. Phosphorylation sites are critical for protein transport and function, so analyzing protein phosphorylation sites helps us to understand the function and regulatory network of proteins comprehensively.

 

Mass spectrometry is a powerful analysis technique. With the development of bioinformatics and proteomics, mass spectrometry has become an important tool for studying protein phosphorylation sites. The commonly used methods are as follows。

 

Methods Based on Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS)

1. Phosphatase Method

After treatment with phosphatase, phosphorylated peptides lose phosphate groups, resulting in specific mass changes. MALDI-TOF-MS determines phosphorylation sites by detecting these changes in mass.

 

2. Phosphopeptide Metastable Ion Metho

Ions generated in the ion source break structurally during their flight in a field-free vacuum tube, losing neutral molecules to produce fragment ions (known as metastable ions). Using a reflectron TOF detector to analyze post-source decay, the appearance of peaks reduced by 98 or 80 can indicate phosphopeptides. If sulfation modifications occur, similar fragment ions are produced, which can be differentiated with the help of phosphatase hydrolysis.

 

3. Positive and Negative Ion Detection

In negative ion detection mode, the response signals of phosphopeptides are stronger than those in positive ion detection mode.

 

Post Source Decay (PSD)

The specific chemical reaction is used to dissociate ions after the ion source of the mass spectrometer, which can generate specific fragment ions containing phosphate groups. These ions present a unique mass-to-charge ratio in the mass spectrum, which can accurately distinguish phosphorylated peptides from non-phosphorylated peptides.

 

Electron Capture Dissociation (ECD)

In ECD-MS, proteins are first fixed on the support film, and the protein dissociates after high-energy ion bombardment to produce a series of fragment ions. These fragment ions contain protein sequence and modification information, including the information of phosphorylation sites.

 

Methods Based on Various Scanning Modes of Tandem Mass Spectrometry

1. Neutral Loss Scan

The core of the neutral loss scan is to monitor the mass of neutral loss, that is, the mass of phosphate group (PO32-), to determine the occurrence of phosphorylation. When the neutral loss scan is activated, the mass spectrometer will pay special attention to those ions that have lost a specific mass (such as the mass of phosphate group, about 98 Da). When the protein dissociates into peptide fragments in the mass spectrometer, if a peptide fragment is phosphorylated, it may lose the phosphate group in a certain dissociation step, forming a neutral molecule. The next-level mass spectrometer will detect this neutral loss and determine the presence of phosphorylation.

 

2. Precursor Ion Scan

Phosphorylated peptides will produce specific fragments of phosphate groups after Collision Induced Dissociation (CID), these specific fragments can serve as "reporter ions" of phosphopeptides when performing precursor ion scanning with tandem mass spectrometry.

 

MtoZ Biolabs uses the latest Obitrap Fusion Lumos mass spectrometer launched by Thermo company in combination with Nano-LC to provide a one-stop service for protein phosphorylation site detection. You only need to tell us your needs and send samples, MtoZ Biolabs is responsible for all subsequent projects, including protein extraction, protein enzymatic hydrolysis, enrichment of phosphorylated peptide segments, peptide separation, mass spectrometry analysis, mass spectrometry raw data analysis, bioinformatics analysis, and provides you with a detailed technical report. Please feel free to consult.