Exosomes are membrane-bound nanovesicles that transport molecular signals (e.g., proteins) between cells and are released from a wide range of cells, including the human placenta. Interestingly, the levels of exosomes present in maternal circulation are higher in preeclamptic pregnancies and their protein content profile change in response to the microenvironment milieu. Through the discovery of candidate biomarkers, mass spectrometry (MS)-based proteomics may provide a better understanding of the pathophysiology underlying pregnancy-associated disorders. With advances in sample preparation techniques, computational methodologies, and bioinformatics, MS-based proteomics have addressed the challenge of identifying and quantifying thousands of proteins and peptides from a variety of complex biological samples. Despite increasing interest in biomarker diagnostics, the complex nature of biological matrices (e.g., plasma) poses a challenge for candidate biomarker discovery. Here, University of Queensland researchers describe a workflow to prepare exosomes for proteomic analysis.
Following the addition of equal volumes of the peptide sample into all wells in the frame, a high voltage is applied to the ends of the gel strip. This causes the peptide molecules to migrate through the gel strip until they are positioned where the pH equals the isoelectric point (pI) of the molecule. The electric field also extends into the liquid phase, where the peptides are suspended. This ensures the molecules remain suspended in solution at their respective pI even after the fractionation run is complete. Do not turn off the fractionator until you are ready to collect the peptide fractions