Labeled Exosomal Proteomic Detection Service

The comparative quantitative analysis of complex protein samples can be through 2DE gel-based proteomics or mass spectrometry (MS)-based methods, using isobaric labeling reagents like isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tag (TMT) to achieve. At present, Creative Biolabs’ proteomics analysis platform has successfully realized the quantitative analysis of exosome samples.

Background of Labeling Proteomics in Exosome

The characterization of exosomal cargo is very important because this cargo can provide clues to the biogenesis, targeting and cellular effects of exosomes, and may be a source of biomarkers for disease diagnosis, prognosis, and response to treatment. With the advent of proteomics technology and the further development of exosome research, the proteomics of exosomes derived from complex organelles and biological fluids has maintained attention as a source of biomarkers for disease status and prediction.

Fig.1 MS has an important role in the characterization of exosomes. (Pocsfalvi, 2016)

So far, a large number of studies have indicated that thousands of proteins may be detected in exosomes, but the number of proteins detected in multiple data sets is much less. In addition, beyond qualitative representation, quantitative composition is essential for any comparison. Among the developed and applied methods of quantitative proteomics, chemical labeling by isobaric tandem mass tags is one of the most popular methods in recent ten years.

Labeling Proteomics Services

In order to be analyzed by MS, the exosomal proteins can be separated by SDS-PAGE, in-gel digested, and extracted peptides for LC-MS/MS experiments. Isobaric peptide labeling strategies are mainly aimed at N-terminal and lysine residues (also cysteine reagents), using N-hydroxysuccinimide reactive groups to promote peptide ammonolysis, replacing the binary adducts composed of mass balance and reporter. Different m/z values come from different isobaric combinations of heavy and light C, N, and O isotopes in alternative forms of reagents, which are detected in tandem MS of pooled labeled samples and used to determine the relative contribution/abundance of peptides, thus determining the relative quantification of proteins.

Fig.2 Schematic illustration of the iTRAQ-based proteomics study. (Song, 2018)

Technical Advantages

iTRAQ technology uses 4-plex or 8-plex isotope labels, while TMT technology uses 6, 10 and 16 isotope labels, which can simultaneously label 2-16 groups of samples for expression difference analysis;
It is an in vitro labeling technology that can label a variety of samples. It can find membrane proteins, nuclear protein, extracellular proteins, strong base and strong acid proteins, and proteins less than 10 kD and greater than 200 kD. It also has a good detection rate for low-abundance protein;
After mixing different labels, it has good parallelism, avoids technical errors, and improves detection efficiency.

As exosomes are found in more and more samples from different sources, especially some samples with relatively precious and low content, the research on their proteomics has become more and more rigorous. In the proteomic analysis of exosome samples, Creative Biolabs insists on continuously developing methods to improve sensitivity, including introducing new chromatography or MS methods, improving exosome sample preparation methods, etc., so as to better serve exosome research. If you are interested in our service, please contact us for more information.

References

Pocsfalvi, G., et al. Mass spectrometry of extracellular vesicles. Mass Spectrometry Reviews. 2016, 35(1): 3-21.
Song, H., et al. Quantitative proteomic study reveals up-regulation of cAMP signaling pathway-related proteins in mild traumatic brain injury. Journal of proteome research. 2018, 17(2): 858-869.

Plant Exosome Isolation