Extracellular Vesicle Proteomics

Exosomes are small vesicles with a lipid bilayer membrane, with diameters ranging from 30 to 150 nm. They are secreted by living cells and are widely present in various body fluids, including blood, saliva, urine, synovial fluid, milk, cerebrospinal fluid, etc. Exosomes are rich in a large number of bioactive molecules, such as nucleic acids, proteins, lipids, and sugars. They serve as important vehicles for transmitting biological information between cells and play important roles in physiological regulation and pathological development, thus becoming a hotspot in recent years.

 

Currently, clinical biological research on miRNAs in exosomes has been very thorough. With the continuous development of mass spectrometry technology, proteins, as the direct executors of many biological functions, are receiving more and more attention, especially the proteins in exosomes and their biological functions. Compared to RNA molecules, protein molecules are more stable and have a more direct impact on intracellular activities. After entering the recipient cells mediated by exosomes, they can immediately exert their activities. The use of mass spectrometry technology in exosomal proteins helps to elucidate the mechanism of disease occurrence and development, and then find specific biomarkers and targets for precision treatment.

 

With the increasing number of exosome-related studies, the research strategy for exosomal proteomics based on mass spectrometry technology is gradually improving. The mass spectrometry technology used for proteomics research is consistent with conventional proteomics research, and can be divided into non-targeted proteomics mass spectrometry technology and targeted proteomics mass spectrometry technology. The non-targeted proteomics mass spectrometry technology detects all proteins within the detection limit in the sample, which is often used for screening or signal pathway analysis of differential proteins in exosomes, including labeled quantitation (in vitro and in vivo) and non-labeled quantitation. The targeted proteomics mass spectrometry technology detects dozens of target proteins, which is often used for the verification of known proteins, including multiple reaction monitoring mass spectrometry (MRM) and parallel reaction monitoring mass spectrometry (PRM), etc.

 

Non-Targeted Proteomics Mass Spectrometry Technology

1. Labeling Quantitative Technology

(1) In vitro labeling quantitative technology

Currently, TMT and iTRAQ are the main in vitro labeling quantitation techniques commonly used in exosomal proteomics research. Both react isotopes with the amine of peptides, and through the principle of isobaric labeling, they label different samples, simultaneously detecting the ion signal response intensity of the same peptide in different samples in the mass spectrometer, achieving the relative quantitation of all proteins. The main difference between TMT and iTRAQ lies in the number of labeled samples. TMT is developed by Thermo Fisher Scientific, suitable for 2, 6, 10, or 16 labels; while iTRAQ is developed by AB Sciex, suitable for 4 or 8 labels.

 

(2) In vivo labeling quantitative technology

SILAC is an in vivo labeling technology. It first labels light, medium, and heavy C/N isotopes on L-lysine and L-arginine, two essential amino acids, then co-cultures the labeled amino acids with cells (>5 generations) or normal animals, achieving >95% protein labeling through normal cell metabolism, suitable for 2 or 3 labels. In the research of exosomal proteomics, SILAC is mainly used for exosomes derived from cell lines. Since no in vitro artificial labeling treatment is needed, it has higher fidelity for protein labeling.

 

2. Non-Labeled Quantitative Technology

The non-labeled quantitative technology compares the peak area or peak intensity of the same peptide in different samples to directly obtain the relative quantitation information of the protein represented by the peptide. Compared with labeled quantitation, non-labeled quantitation can be used for any type of sample and does not require complex labeling technology in advance. By eliminating the labeling step, errors caused by the labeling are reduced. Moreover, since each sample is analyzed separately, the trouble of analyzing the number of samples is solved, and even high-throughput quantitation analysis of multiple biological samples can be realized simultaneously.

 

Targeted Proteomics Mass Spectrometry Technology

1. Multiple Reaction Monitoring Mass Spectrometry (MRM)

The principle of MRM is to set the parent ion information of the target protein in the first mass spectrometry, and set the corresponding daughter ion information in the second mass spectrometry. By selectively collecting the signal of the parent ion-daughter ion pair, the relative quantitation of the target protein can be realized.

 

2. Parallel Reaction Monitoring Mass Spectrometry (PRM)

In 2012, PETERSON proposed the use of high-resolution mass spectrometers (orbitrap/TOF) to replace the third quadrupole mass analyzer in the triple quadrupole to collect all daughter ion information, and then quantitate the target protein, namely PRM. With the maturity of technology and the improvement of methodology, PRM has been increasingly used in the research of exosomal proteomics. PRM is derived from MRM, combining the high selectivity of quadrupoles and the high resolution and accuracy of Orbitrap. After the selected parent ions are cleaved, all fragment ions are measured, which has better detection sensitivity and anti-interference ability.

 

At present, with the increasing diversity of diseases, traditional diagnostic and therapeutic methods can no longer meet people's expectations for disease diagnosis and treatment. Precision medicine that emerged in response has become a hot topic. Exosomal proteomics represents specific markers of cells and can reflect the state of cells to the greatest extent, thus becoming potential candidates for disease diagnosis and treatment. Due to its high sensitivity, high specificity, and wide range of analysis, mass spectrometry technology has gradually become the main research method in proteomics. MtoZ Biolabs uses the latest Obitrap Fusion Lumos mass spectrometer from Thermo, in combination with nanoLC-MS/MS nanoscale chromatography, to perform efficient and accurate identification and analysis of exosomal proteomics. In addition, we provide one-stop service, from the separation of exosomes in the early stage to the detection of biomarkers in the later stage, we all have professional personnel, welcome your consultation.