Proteomic Analysis of Heat Proteins

Thermal Proteome Profiling (TPP) is a proteome-wide technology introduced in 2014, which essentially combines Cellular Thermal Shift Assay (CETSA) with Quantitative Mass Spectrometry (MS) to detect the solubility curves of thousands of expressed proteins. This method is widely used for the identification of drug target proteins and off-target proteins.

 

Essentially, the principle of TPP is similar to that of CETSA, both based on the fact that protein denatures under heat will gradually unfold and precipitate; the difference is that TPP uses multi-tagged mass spectrometry technology for protein quantification, while the original CETSA uses an antibody-based quantification technology. Therefore, this thermal shift technology based on high-throughput mass spectrometry for protein quantification is also known as MS-CETSA.

 

The Primary Five Steps in TPP

1. Sample Preparation

It includes the preparation of cell materials and condition changes. Cell materials can be cell extracts, whole cells, tissues, body fluids, and microorganisms, etc.; condition changes are usually chemical (such as drugs or metabolites), genetic (such as gene knockout or overexpression), environmental changes and different cell states (such as different stages of the cell cycle), and it can also study the melting behavior of proteins in situ without condition changes.

 

2. Heat Treatment

The sample is subjected to short heat treatment to induce protein denature aggregation, usually for 3 min.

 

3. Collection of Soluble Proteome Components

The supernatant is collected by ultracentrifugation or the soluble proteome components are collected by low-speed centrifugation using a porous filter plate.

 

4. Mass Spectrometry-Based Proteome Analysis

The soluble proteins are digested into peptides with protease, then different samples are labeled with Tandem Mass Tag (TMT) by using stable isotope labeling reagent, and finally the abundance of peptides in each sample is quantified by mass spectrometry.

 

5. Data Analysis

The raw mass spectrometry data obtained are processed with a proteomics database to identify and quantify the detected proteins.

 

Originally developed for finding drug molecule targets and off-targets, TPP has seen its applications expand over recent years. Today, TPP can be used to study post-translational modifications, interactions between proteins, interactions between nucleic acids and proteins, and basic protein functions, to reveal the mysteries of metabolic pathways. This widespread application of the technique brings new perspectives and possibilities to the fields of biomedical research and drug development.

 

MtoZ Biolabs uses the ThermoFisher OrbitrapFusionLumos mass spectrometry platform combined with nanoLC-MS/MS nanoscale chromatography, to provide you with a one-stop technical package for efficient and accurate thermal proteomics services. It can monitor the melting curves of thousands of expressed proteins, assist in the identification of drug targets and off-targets, or studies on protein-metabolite and protein-protein interactions. Feel free to consult.