Technical introduction

Large-scale qualitative and quantitative analysis of thousands of proteins in biological samples can be performed simultaneously by whole proteome extraction, enzymatic hydrolysis, peptide classification, high-resolution mass spectrometry data collection and database retrieval. Compare the types and abundance of proteins in cell or tissue samples in sickness and health, or before and after drug treatment, to find targets of drug action. We provide standardized sample processing protocols, qualitative and quantitative identification of proteomics and professional data analysis strategies.

Service content

1. Quantitative analysis of whole-proteome differential proteins caused by diseases, drug treatment and environmental stimulation

2. Quantitative analysis of whole proteome of subcellular structure (cell membrane, nucleus, mitochondria, etc.)

3. Discovery of whole-proteome level biomarkers

Proteome quantitative analysis techniques:

Non-label quantitative technique (LFQ) :

Technique: The protein content of different samples can be analyzed by comparing the number of spectra or the area of ion peaks

Advantage: Rapid comparison of protein abundance differences between large numbers of samples without isotope labeling

Application: Cell, tissue, blood and other samples

Reduction dimethylation labeling Quantitative Technique (REDI) :

Technique: The quantitative comparison of proteins between two or three samples can be realized by using isotope tags and the reaction of lysine side chain and N-terminal amino group of peptide

Advantages: economical and convenient, strong universality

Application: Cell, tissue, blood and other samples

Quantitative Labeling of Stable Isotope Amino Acid Metabolism (SILAC) :

Technique: Proteome quantification between different samples was achieved by cultured cells with stable isotopes in medium

Advantages: Less influence of operating error, more accurate quantitative

Application: Living cell samples

Tandem mass spectrometry labeled Quantitative Technique (TMT/IBT) :

Technique: Using TMT/IBT reagent to label the peptide, detect the signal strength of the reporter group in the sample, and realize the quantitative comparison of proteins in different samples

Advantages: Up to 16 samples can be analyzed, quantitative accuracy

Application: Cell, tissue, blood and other samples

Case presentation

Technical background

Proteins are the agents of biological activity and the main targets of drug development. Evaluating drug effects at the proteome level is crucial to the detection, validation and optimization of drug targets, and also greatly promotes the development of innovative drugs. Target discovery based on differential proteomic analysis usually uses the strategy of comparing protein expression differences in cells or tissues before and after disease and normal physiological state or drug treatment to find targets of drug action.

Case analysis

Project requirements: Comparison of drug treatment groups and control groups, whole-proteome level changes, and exploration of molecular mechanisms related to drug phenotypes

Sample type: Drug and control cell samples with three biological replicates per group

Solution: A quantitative proteomics method based on TMT multiple isotope labeling technique was used to quantitatively identify proteins with different total protein levels

Data presentation:

As shown in the volcanic map of protein abundance difference, there were 5,987 common quantitative proteins in the six groups of samples, and significance test analysis was conducted on the ratio of each protein. The abundance of drug-treated histones was up-regulated to 560, and down-regulated to 363, and relevant intensity information was also displayed in the form of heat map.

The different proteins were analyzed by Gene_ontology, including GOTERM_ Biological process. GOTERM_ Cellular component; GOTERM_ Molecular function, KEGG_pathway, by evaluating the significance level of GO term enrichment to find the functional classification and pathway of significant enrichment of differential proteins, thus contributing to the judgment of drug molecular mechanism.

As shown above, upregulated proteins are significantly enriched in nuclear chromosome segregation, mitotic sister chromatid segregation, sister chromatid segregation and other signaling pathways. The results indicated that the drug affected the process of chromatin separation at the molecular level.