Advantages and Disadvantages of Methods for Detecting Protein Phosphorylation

Protein phosphorylation is one of the most important post-translational modifications of proteins, and phosphorylation occurs in about 1/3 of all proteins in eukaryotic cells. Catalyzed by protein kinases, phosphate groups are transferred from the donor molecule to the side chains of amino acids containing hydroxyl groups of the protein. Protein phosphorylation is reversible, and protein phosphorylation and dephosphorylation regulate almost all processes of life activity. Exploring the mystery of the phosphorylation modification process and its impact on life activities has become an issue of interest to many biochemistry and proteomics researchers. The identification and detection of phosphorylated proteins is one of the keys to proteomics research. So, what are the detection methods for protein phosphorylation?

Common methods for protein phosphorylation detection

Western Blot

Phosphorylation of proteins can be detected by western blot using antibodies against phosphorylated proteins. It uses polyacrylamide gel electrophoresis with primary antibodies as probes, combined with labeled secondary antibodies, to show protein bands by autoradiography or substrate color development. After passing through the concentrated and separated gel, the protein samples are arranged on the gel surface by molecular weight size.Then, the protein is transferred to the membrane, where it will be firmly bound to the carrier in the form of non-covalent bonds, react with the corresponding phosphorylated antibody, then interact with the enzyme or isotope-labeled secondary antibody, and finally detect the specific phosphorylated protein component separated by electrophoresis.

Advantages: good specificity and high resolution.

Disadvantages: proteins with weak phosphorylation signals in cells need to be enriched before they can be detected. And analysis and preparation can not use the same piece of gel, sometimes the detected protein spots are difficult to correspond with the protein spots on the gel, which is not conducive to accurate analysis.

Suitable for single protein phosphorylation detection

32P isotope radiolabeling

During metabolic culture in vivo, 32P isotope radiolabeled phosphate is used as a phosphate group donor, and after phosphorylation enzymatic reaction, the 32P isotope radiolabeled phosphate group is transferred to the corresponding reactive protein. Phosphorylated proteins are separated by gel electrophoresis and can be detected by autoradiography.

Advantages: It can provide the exact phosphorylation site, and the operation is simple.

Disadvantages: Radioactive labels can only be applied in cells and are radioactive.

Suitable for protein phosphorylation site detection and single protein phosphorylation detection

Flow cytometry

Flow cytometry uses a fluorescently labeled single cell suspension as the assay sample and determines the concentration of the cell or cytokine in question by detecting the fluorescence intensity. Flow cytometry detects specific antibodies to fluorescently labeled phosphorylated proteins, allows precise measurement of phosphorylation in single cells, and allows simultaneous analysis of several components of signaling pathways in different cell subpopulations.

Advantages: it can detect phosphorylation levels in various cell subpopulations, explore signal transduction mechanisms, and allow simultaneous analysis of the interrelationships between multiple parameters.

Disadvantages: specific fluorescent dyes are required?high cost and expensive instruments.

Suitable for multi-parameter phosphorylation assays at the single-cell level

Mass spectrometry can determine the primary structure of proteins, including molecular weight, amino acid sequence and the number of peptides or disulfide bonds and their sites, and is a method that can detect and identify post-translational modifications. Protein phosphorylation mass spectrometry detection includes: identification of phosphorylated peptides; determination of phosphorylation type based on amino acid sequence; and identification of phosphorylation sites.

Advantages: it can discover and determine the primary structure of phosphorylated proteins, determine the phosphorylation sites of proteins, and has excellent sensitivity and resolution in identifying individual proteins.

Disadvantages: Mass spectrometry analysis of enzymatically cleaved phosphorylated proteins does not detect the entire protein sequence and there may be important phosphorylated regions that are overlooked.

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