Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry.
Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.
Kang
Dukjin
D
Department of Chemistry, Yonsei University, Seoul, Korea.
Oh
Sunok
S
Ahn
Sung-Min
SM
Lee
Bong-Hee
BH
Moon
Myeong Hee
MH
D002478:Cells, Cultured; D002853:Chromatography, Liquid; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D031521:Fractionation, Field Flow; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles; D009474:Neurons / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013234:Stem Cells / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D008565:Membrane Proteins; D020543:Proteome