一种基于蛋白质组学的分离分析外泌体蛋白质

Through the discovery of candidate biomarkers, mass spectrometry(MS)-based proteomics may provide a better understanding of the pathophysiology underlying disease. Despite increasing interest in biomarker diagnostics, the complex nature of biological matrices such as plasma pose a challenge for candidate biomarker discovery. In this paper, the author describe a workflow to prepare exosomes for proteomic analysis.

Proteomic analysis of complex secretomes can be further improved by fractionation at the peptide or protein level. This paper will focus on off-gel isoelectric focusing, where peptides are fractionated according to their isoelectric point along an immobilized pH gradient strip.This liquid-phase recovery of peptides fractions makes off-gel fractions directly compatible with liquid-phase workflow such as liquid chromatography-tandem mass spectrometry(LC-MS/MS). Thus, the primary aim of this paper is to established a robust EV preparation protocol for proteomic profiling within the context of a bottom-up proteomics workflow.

Workflow：

Desaling:

Fig punch out the empore c18 membrane using a cut down 200ul pipette tip in a sterile petri dish or surface

Fig transfer the membrane to a gel-load tip using a needle or a another gel-load tip ensuring the membrane is compressed down into the final stage coloum with on spaces.

Off-gel fractionation:

Fig following the addition of equal volumes of the peptide sample into all wells inthe frame, a high voltage is applled 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 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 collected the peptide fractions.