Protein Phosphorylation Modification Types, Enrichment and Identification Methods

In the life cycle of a mammalian cell, about one-third of proteins are phosphorylated. In vertebrate genes, about 5% of genes encode proteins are protein kinase and phosphatase involved in phosphorylation and dephosphorylation. It can be involved in regulating the growth, development, response to stress, disease occurrence and other life processes by stimulating and regulating many signaling pathways. It has always been the focus and hotspot of biological research.

Protein Phosphorylation

Protein phosphorylation refers to the process of promoting protein post-translational modification, which transfers ATP or GTP phosphate group (P04) to different kinds of amino acids (mainly include serine S, threonine T, tyrosine Y) under the catalysis of kinase.

Types of Phosphorylation Modification

Phosphorylated proteins can be classified into four classes depending on the amino acid residues of the phosphate, namely, O-phosphate protein, N-phosphate protein, acyl phosphate protein, and S-phosphate protein.

O-phosphate protein is formed by phosphorylation of hydroxy amino acids such as serine, threonine or tyrosine. N-phosphate protein is formed by phosphorylation of arginine, lysine or histidine. Acyl phosphate protein is formed by phosphorylation of aspartic acid or glutamic acid. S-phosphate protein is formed by cysteine phosphorylation.

Protein Phosphorylation Identification Methods

Western Blotting

Western Blotting is an immunobiochemical analysis method based on gel electrophoresis and solid phase immunoassay technology to detect a certain protein in a complex sample according to specific antigen-antibody specific reaction.sdfs

Technical Advantages

• High detection specificity

• Ability to specifically identify individual phospho site
• Phosphorylation is limited by phosphorylated antibodies, therefore highly specific and specific phosphorylated antibodies are difficult to prepare. While, newly discovered phosphorylation sites often lack effective phosphorylation detection antibodies.

Isotope-labeled Western Blotting

The principle of this method is to identify the gelshift caused by the incorporation of radioactive 32P and the change of molecular mass when the protein is phosphorylated, and then detect the isotope label by Western Blotting to achieve the purpose of identifying the phosphorylated protein.

Technical Advantages

• Detection technology is sensitive and intuitive
• Commonly used for in vitro phosphorylation reaction detection
• Phosphorylation is not restricted by antibodies
• Simultaneous analysis of multiple protein phosphorylation

Mn2+-Phos-tag SDS-PAGE

Mn2+-Phos-tag Sds-Page allows the retention of phosphorylated proteins by the affinity of the dinuclear metal (such as Mn2+) complex attached to the acrylamide molecule to the phosphate group, and then transfers the electrophoresis results to the PVDF membrane. Protein recognition by the corresponding protein was used to detect protein phosphorylation based on migration retention.

Technical Advantages

• No radiation hazard
• Identification is not limited to antibodies that specifically recognize protein phosphorylation
• Suitable for large-scale phosphorylated protein analysis
• Compatible with mass spectrometry for more detailed analysis and identification

Mass spectrometry Method

In mass spectrometry, the components in the sample are ionized to generate ions of different charge-to-mass ratios, and then the ion beam is generated by the action of the accelerating electric field, and enters the mass analyzer. In the mass analyzer, under the action of a magnetic field, ions with the same mass-to-charge ratio are focused at the same point, and ions of different mass-to-charge ratios are focused at different points, thus obtaining a mass spectrum that distinguishes ions of different mass-to-charge ratios. Compared to unmodified peptides, phosphate group-modified peptides increase by 79.983 in relative molecular mass, thereby enabling differentiation from unmodified peptides in the mass spectrum.

pY-1000 Motif Antibody Enrichment

High-affinity antibodies can immunoprecipitate specific proteins from complex mixtures and selectively separate phosphorylated proteins. Among them, the monoclonal antibody of tyrosine phosphorylated protein is a known antibody for detecting phosphorylated protein, which has strong affinity and can effectively immunoprecipitate tyrosine phosphorylated protein. Moreover, the relative proportion of tyrosine phosphorylation is extremely low, accounting for only 1% of the phosphate group, but it is the most important in the field of tumors and cell signal transduction. It is difficult to enrich with the above TiO2 and IMAC methods. Currently, immunoprecipitation of tyrosine phosphorylation at the peptide level using motif antibodies is the preferred method for analyzing this phosphorylation.

Prime Jones is a senior researcher from MtoZ Biolabs. She is specialized in the field of proteomics study.