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Methods Uniquely Developed for Exosome Mass Spec Analysis

SBI routinely generates high quality exosome peptide libraries for Mass spec analysis. The Mass Spec service works with exosomes isolated using ultracentrifugation as well as using ExoQuick (serum, plasma, ascites samples) or ExoQuick-TC (cell media, urine, spinal fluid) or immunopurified for specific exosome subpopulations using SBI's Exo-Flow IP kits. The MS service offers the choice of creating either exosome surface protein "shaving" peptide libraries for surface marker discovery or complete exosome peptide library preparations. SBI's exosome Mass Spec service can be ordered using catalog# CSEQ810A-1.

What is exosome "shaving" versus "complete" peptide libraries?

   

   shaving or complete peptide libraries

Sample XPEP-Shaved exosome NanoSight data

Exosome surface protein content can be a rich source of biomarkers as well as potentially identifying a good surface marker for downstream antibody-bead purification and FACs experiments. In this example, the exosomes were first isolated from the tissue culture medium from HEK293 cells grown in Exo-FBS exosome-depleted media supplement with standard DMEM. The cells were grown to 80-90% confluency in a 10 cm cell culture dish. The secreted exosomes were isolated using ExoQuick-TC. The exosome pellet was processed using the XPEP Shaving procedure. The shaved exosomes remain intact, but lose their surface protein "coats" and their diameters condense into a more homogeneous population.

shaving exosomes and nanosight data

 

Sample MS peptide library SDS-PAGE data

   peptide SDS-PAGE profiles

Exosome Mass Spec protein libraries prepared by either the Shave or Complete protocols produce ideal peptide libraries for MS/MS analysis. Approximately 10 ug of the peptide libraries were separated on a 4-20% gradient SDS PAGE gel and stained with Imperial blue to visualize the library peptide size distributions. The peptide library fragments generated are of the expected 2-10 kD fragment range and optimal for Mass Spec analysis.

Exosome protein profiles to expect

   what to expect

The protein content of exosomes can vary greatly depending upon the cell source from which it originated. Some common surface and internal proteins can be observed in typical exosomes and are identified in Mass Spec data. Some common proteins to look for include: HSP (Heat Shock) proteins, GAPDH, Keratins, Tubulin, Actin, Vimentin, Fibulin, Fibronectin, Annexins, Flotillins, Galectin and α-Enolase. The figure to the right from: David G. Meckes Jr. and Nancy Raab-Traub, "Microvesicles and Viral Infection". J. Virol. December 2011 vol. 85 no. 24 12844-12854.

Sample XPEP serum exosome MS data

Exosomes were isolated from 500ul control human serum using ExoQuick. The exosome pellet was processed using the XPEP Complete procedure. The peptide libraries were then analyzed by nano LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded on a trapping column and eluted over a 75um analytical column at 350nL/min using a 2hr reverse phase gradient; both columns were packed with Jupiter Proteo resin (Phenomenex). The injection volume was 30uL. The mass spectrometer was operated in datadependent mode, with the Orbitrap operating at 60,000 FWHM and 17,500 FWHM for MS and MS/MS respectively. The fifteen most abundant ions were selected for MS/MS and data analyzed using MASCOT databases and Scaffold software.

The table below shows some example serum exosome protein MS/MS data with common exosome proteins highlighted in yellow.

sample exosome MS data table