Quantitative Phosphoproteomic Analysis

It is estimated that about 1/3 of proteins can be phosphorylated at any time in a living organism. Identification of phosphorylated proteins and understanding the dynamics of protein phosphorylation, including the phosphorylation or dephosphorylation of a specific amino acid residue in response to cellular and environmental factors, can help to study the regulation of biological networks at a global level. This emerging field of systems biology is known as phosphoproteomics. Comprehensive analysis of all phosphorylated proteins in a biological system, including identification of all phosphorylated proteins, precise localization of phosphorylation sites, and quantification of differentially expressed phosphorylated proteins, is termed phosphoproteome analysis. Quantitative analysis of phosphoproteomics helps us to better understand cellular signaling mechanisms, discover disease biomarkers, drive drug development, and achieve precision treatment. Several methods have been established to quantify changes in protein phosphorylation, some of which include:

 

Isotope Metabolic Labeling Techniques

1. 15N Labeling

Cells are cultured separately in two types of media, one of which uses 15N as the nitrogen source. After mixing cells from the two types of media and extracting and isolating the target protein, the protein is enzymatically digested and then analyzed by mass spectrometry. The 14N/15N isotope abundance ratio of peaks can reflect the relative expression levels of proteins from the two sources, and the degree of phosphorylation can be quantified relatively based on the ratio.

 

Features: This method is very suitable for tracking the dynamic changes of a single phosphorylation site, but it is limited to the analysis of proteins with relatively high expression levels. The target protein needs to be isolated and purified, which is not suitable for large-scale protein quantification.

 

2. SILAC Labeling

After the appearance of SILAC technology, it quickly replaced 15N labeling. This technique uses culture media containing light and heavy isotope-labeled amino acids to label cells separately, resulting in stable isotope labeling of intracellular proteins. Protein extracts from cells are mixed in equal amounts, digested by enzymes, and then identified by mass spectrometry. The relative amounts of different labeled peptide segments are determined by analyzing the relative amounts of different labeled peptide segments. In addition, technologies such as iTRAQ and iCAT, which can be used for peptide quantification, can also be used for the study of phosphorylated peptides.

 

Features: This technique has the advantages of low protein loss, high labeling efficiency, low error, the ability to perform multiple sample comparisons, and high peptide coverage. However, this method can only be used for ex-vivo cell labeling and is relatively expensive.

 

3. 18O Labeling

The principle is to put the protein in H218O during or after enzymatic digestion. Under the catalysis of protease, the two oxygen atoms on the carboxyl group are replaced by 18O. Except for the C-terminal peptide, all peptide segments can generally be replaced with 2 18O under suitable conditions, resulting in a mass difference of +4 u for the peptide segments.

 

Features: It often produces the phenomenon of marking one oxygen atom and two oxygen atoms unevenly, and the area resolution of the ion chromatogram (EIC) is low and overlapping peaks appear.

 

Chemical Labeling Techniques

1. Phosphopeptide Isotope Affinity Labeling (PhIAT)

In recent years, isotope-coded affinity tag (ICAT) technology has been widely used to quantify protein expression levels and has been further extended to quantify phosphorylation levels. The basic principle is that the phosphate group of phosphorylated peptides undergoes β-elimination in an alkaline environment to form a double bond, and a nucleophilic reagent labeled with different isotopes reacts with the double bond to replace the phosphate group.

 

Features: This method is suitable for quantifying peptides containing phosphoserine and phosphothreonine residues. However, the efficiency of chemical reactions is generally low, and phosphorylation only occurs in a small part of the whole protein, which presents certain difficulties in the quantitative analysis of whole protein phosphorylation.

 

2. Affinity Chromatography Combined with Isotope Labeling

Different methylation reagents (methanol and deuterated methanol) are used for the two samples. After the phosphorylated peptides are enriched by affinity chromatography, they are analyzed by LC-MS. The abundance ratio of paired phosphorylated peptides is used for the quantitative study of phosphorylated peptides.

 

Features: It is difficult to confirm paired phosphorylated peptides with this technology and special software analysis is needed. The mass difference of paired phosphorylated peptides is not consistent and is related to the number of residues that can be methylated in the peptide segment. At the same time, some glutamine residues undergo deamination to become glutamic acid, which can also be methylated, increasing the difficulty of analysis.

 

3. Pro-Q Diamond Phosphoprotein Staining

The principle is to use a specific fluorescent dye to react with the phosphate group in phosphorylated proteins, thereby staining phosphorylated proteins. The fluorescent signal on the gel is then detected by a fluorescence scanner to achieve quantitative detection of phosphorylated proteins. This process can be performed directly on sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and two-dimensional gels (2D-PAGE).

 

Features: This technique is particularly suitable for the detection of protein kinase and phosphatase subunits. This method of staining is fast, simple, reversible, and easy to operate, and is compatible with various proteomics methods. However, there is non-specific staining, and the fluorescent signal is easily affected by various factors.

 

MtoZ Biolabs uses Thermo Fisher's Q ExactiveHF mass spectrometry platform, Orbitrap Fusion mass spectrometry platform, Orbitrap Fusion Lumos mass spectrometry platform combined with Nano-LC, and offers a package of phosphoproteome quantitative analysis services. You just need to tell us your experimental purpose and send us your samples. MtoZ Biolabs will take care of everything else, including protein extraction, protein digestion, enrichment of phosphorylated peptide segments, peptide separation, mass spectrometry analysis, analysis of raw mass spectrometry data, and bioinformatics analysis.