Mass Spectrometry Phosphorylation

Protein phosphorylation is a post-translational modification catalyzed by protein kinases and phosphatases, playing a crucial role in many cellular processes.

 

The Importance of Protein Phosphorylation

1. Regulating Protein Stability

It controls the stability of proteins by modifying their degradation rates.

 

2. Mediating Protein Activity

It either activates or inhibits the activity of proteins by changing their conformations.

 

3. Involvement in Cell Signaling

Through phosphorylation, cells can respond to external stimuli, regulating cellular processes such as growth, differentiation, and apoptosis.

 

4. Regulating Cellular Physiological Functions

This includes metabolism, transcription regulation, translation regulation, protein degradation, cellular dynamic balance, cell proliferation, cell differentiation, and cell survival.

 

Mass spectrometry is a common analytical technique in biological research in recent years. It can determine the primary structure of proteins, including molecular weight, peptide chain amino acid sequencing, and the number and location of disulfide bonds, and is a method for the discovery and identification of post-translational modifications. The steps for detecting protein phosphorylation using mass spectrometry include: ①enrichment of phosphorylated peptides; ②detection of phosphorylated peptides and determination of phosphorylation sites; ③quantitative research of protein phosphorylation; ④analysis of mass spectrometry data.

 

Enrichment of Phosphorylated Peptides

Only a small part of the proteome is phosphorylated. Phosphorylated peptides often appear at low abundance in protein hydrolysis products, and the response of mass spectrometry to phosphorylated peptides is often interfered with by high-abundance non-phosphorylated peptides. Therefore, when the obtained protein content is extremely limited, the content of some non-phosphorylated peptides needs to be reduced (or enriched phosphorylated peptides) before analyzing phosphorylated peptides with mass spectrometry. Common enrichment methods include immunoprecipitation and immobilized metal affinity chromatography (IMAC).

 

Detection of Phosphorylated Peptides and Determination of Phosphorylation Sites

After sample preparation, phosphorylated peptides need to be detected and the phosphorylation sites of proteins further determined. Usually, a combination of peptide mass fingerprinting (PMF) identification based on MALDI-TOF MS and phosphatase treatment is used to determine phosphorylation sites. After treatment of phosphorylated peptides with phosphatase, the mass-to-charge ratio of peptide segments will undergo specific changes. By detecting these changes in the mass-to-charge ratio with MALDI-TOF MS, phosphorylation sites can be determined.

 

Quantitative Study of Protein Phosphorylation

The identification of phosphorylated amino acid residues in proteins is only the first step to understand signal transduction based on phosphorylation changes. It is necessary to quantify the changes in protein phosphorylation in response to specific stimuli, to describe the relationship between the phosphorylation of specific residues of a protein and how it affects protein function. The quantitative methods for proteomics based on mass spectrometry can be divided into two major categories: labeled quantitation and unlabeled quantitation. The methods commonly used for protein phosphorylation quantitation research are mainly stable isotope labeling (SILAC) and metal element labeling, which are labeled quantitation methods. In addition, technologies such as iTRAQ and iCAT can also be used for the quantitative study of phosphorylated peptide segments.

 

Analysis of Mass Spectrometry Data

The data obtained from mass spectrometry can only be interpreted after database retrieval, and the database is an important guarantee for the analysis of mass spectrometry database retrieval methods. For model organisms and species with many known protein sequences, mass spectrometry data analysis is usually simpler, and the results obtained are reliable and have a high success rate. If the known protein sequences of the studied species are less or incomplete, the method of cross-species retrieval to retrieve the database of related species is usually used, and this method has low identification efficiency and poor accuracy. SwissProt and TrEMBL are common and important protein databases, containing detailed information about all currently known proteins. In addition, there are other databases like NCBInr and Mascot.

 

MtoZ Biolabs uses Thermo's latest Orbitrap Fusion Lumos mass spectrometer combined with Nano-LC to provide researchers with one-stop protein phosphorylation detection services. Simply let us know your requirements and send us your samples. MtoZ Biolabs takes care of all subsequent phases of the project, including protein extraction, protein digestion, enrichment of phosphorylated or glycosylated peptides, peptide separation, mass spectrometry analysis, mass spectrometry raw data analysis, all bioinformatics analysis, and provides you with detailed technical. Feel free to inquire.