How to Detect Protein Phosphorylation?

Protein Phosphorylation is the most common and widely studied form of post-translational protein modification in organisms. It is the process in which a kinase catalyzes the transfer of a phosphate group from ATP or GTP to an amino acid residue of a substrate protein. This process affects the function of over one-third of proteins in human cells.

 

The reversible process of protein phosphorylation and dephosphorylation regulates nearly all life processes, including cell proliferation, development, differentiation, signal transduction, cell apoptosis, neural activity, muscle contraction, and tumor occurrence. Hence, its detection is particularly crucial. The commonly used methods for detecting protein phosphorylation modifications are as follows:

 

32P Isotope Radioactive Labeling Method

The 32P radioactive labeling method is the most classic method for detecting phosphorylated proteins. It principally involves in vitro labeling through purified kinase and [γ-32P], or in vivo labeling by balancing intracellular ATP levels with [γ-32P]-ATP or 32PO43- (orthophosphate). The proteins are then separated using techniques such as one-dimensional or two-dimensional gel electrophoresis (1- or 2-DE), and phosphorylated proteins are detected using radiography or phosphor screens.

 

Western Blot

After running protein samples on SDS-PAGE, clear bands arranged based on molecular weight form on the gel surface. These proteins are then transferred to PVDF (polyvinylidene fluoride membrane) or NC membrane (nitrocellulose membrane) and bound with phospho-specific antibodies. Detection is done by using enzyme or isotope-labeled secondary antibodies, and phosphorylated proteins are observed through color development or autoradiography.

 

Fluorescence Staining Method

Pro-Q Diamond is a fluorescent dye specifically designed to label phosphorylated proteins. Fluorescent scanning can directly display the separated phosphorylated proteins on one-dimensional or two-dimensional gel electrophoresis gels, with low reactivity to non-phosphorylated proteins. The fluorescence intensity changes quantitatively with varying degrees of protein phosphorylation. This dye can be used in conjunction with other fluorescent dyes that detect total protein to simultaneously display the total protein and phosphoprotein spectra on the gel.

 

Flow Cytometry

Flow cytometry uses fluorescently labeled single-cell suspensions as test samples, and the concentration of the tested cells or cellular factors is determined by detecting the fluorescence intensity of the labeled fluorescence. Using flow cytometry to detect fluorescently labeled phosphoprotein-specific antibodies can accurately measure multiple parameters of phosphorylation in single cells, and analyze several components of signaling pathways in different cell subpopulations, significantly increasing the workload.

 

Mass Spectrometry

The principle of mass spectrometry involves ionizing protein peptides. Because of different mass-to-charge ratios, peptides separate when entering the analyzer. The protein is identified and functionally analyzed by measuring the related parameters of the separated peptide ions and using software. Phosphorylated protein peptides are often unstable and spontaneously decompose into metastable states or undergo collision-induced dissociation in the mass spectrometer. Phosphopeptides are prone to lose H3PO4 or HPO3 after treatment with phosphatase. Phosphoserine or phosphothreonine loses 98 u in mass number, while phosphotyrosine loses only 80 u due to the phosphate group being connected to the benzene ring. Mass spectrometry utilizes these features to identify phosphopeptides.

 

Bio-Plex Suspension Array

The Bio-Plex suspension array system is a powerful chip technology platform provided by Bio-Rad. This technology integrates a software package, system verification tools, microsphere coupling reagents, and ready-to-use cytokine and phosphoprotein test kits, enabling simultaneous analysis of various biomolecules (including enzymatic substrates, receptors, antigens, or antibodies) in a single sample. The data obtained from multiplex detection with a suspension array help reveal the interrelationships between biomolecules and signaling pathways as a whole.

 

Identifying the phosphorylation modifications of relevant proteins in protein complex samples is an important part of the proteomics field. There are now various protein phosphorylation analysis methods, each with its advantages and disadvantages, that can be selected based on experimental needs. MtoZ Biolabs offers one-stop protein phosphorylation detection service by using Thermo's latest Orbitrap Fusion Lumos mass spectrometer in combination with Nano-LC. Just let us know your needs and send your samples, and MtoZ Biolabs will take care of all subsequent tasks, including protein extraction, protein enzymatic digestion, enrichment of phosphorylated or glycosylated peptide segments, peptide separation, mass spectrometry analysis, analysis of raw mass spectrometry data, bioinformatics analysis, and more. Feel free to contact us for more information.