Proteomic Analysis of Exosomes from Human Neural Stem Cells by Flow Field-Flow Fractionation and Nanoflow Liquid Chromatography-Tandem Mass Spectrometry Dukjin Kang, † Sunok Oh, † Sung-Min Ahn, ‡ Bong-Hee Lee,* ,‡ and Myeong Hee Moon* ,† Department of Chemistry, Yonsei University, Seoul, 120-749, Korea, and Center for Genomics and Proteomics, Lee Gil Ya Cancer and Diabetes Center, Gachon University of Medicine and Science, Incheon, Korea Received March 26, 2008 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. Keywords: flow field-flow fractionation • FlFFF • protein separation • exosome • proteomics • nanoflow LC-ESI-MS-MS • mass spectrometry • size separation of exosomes Introduction Exosomes are small membrane vesicles (30-100 nm in diameter) secreted by a multitude of cell types as a conse- quence of the fusion of multivesicular late endosomes/lysos- omes with the plasma membrane. 1–3 However, the precise biological functions of exosomes are not fully understood and are dependent on their cell of origin. Although exosomes were first found as a mechanism for shedding membrane proteins such as transferrin receptors during the maturation of reticu- locytes, 4 it is now believed that they are involved in a wide range of physiological functions such as intercellular com- munication, membrane exchange between cells, and as an alternative to lysosomal degradation. 5 Regardless of their putative physiological roles, exosomes also have the potential to be used for cancer immunotherapy. Wolfers and colleagues 6 have suggested that exosomes represent a novel source of tumor-rejection antigens for T-cell cross priming, relevant for immunointervention. In addition, exosomes from dendritic cells (DCs) pulsed with tumor-peptides were able to induce antigen-specific T-cell-mediated immune responses in mice. 7 Also, it was recently reported that, in stem cells, exosomes can deliver not only proteins but also mRNAs, both of which mediate reprogramming of recipient cells. 8 Exosomes are stable at high temperature with a density range from 1.13 to 1.21 g/mL by density gradient centrifugation (sucrose/ D 2 O), and can be recognized by expression of a few characteristic proteins (i.e., endocytic markers, tetraspanins and hsp73). 9 The overall size of exosomes has been an important criterion for distinguishing them from other exovesicles, since eukaryotic cells also secrete membrane vesicles directly from the plasma mem- brane with a mechanism similar to that of viral budding. Mem- brane vesicles are relatively large and heterogeneous in size ranging from 100 to over 1000 nm. 10 However, it is not easy to analytically distinguish exosomes from membrane vesicles due to the limitations of common separation procedures, including ultracentrifugation and flow cytometry. Heijnen and colleagues 11 used flow cytometry to analyze microvesicles and exosomes released from activated platelets, and found that exosomes were too small to be detected by flow cytometry, thus, making them indistinguishable from microvesicles. In contrast to flow cytometry, flow field-flow fractionation (FlFFF), an elution-based technique, can separate and char- acterize macromolecules (e.g., proteins) and nano- to micron- sized particles (e.g., organelles and cells). 12–18 In FlFFF, sepa- ration is carried out in a thin and empty rectangular channel with migration flow moving along the channel axis, while sample retention is controlled by the rate of a secondary flow (crossflow in FlFFF) that is applied across the thin channel through a porous channel wall. In the FlFFF channel, there is a balance between the driving force and the diffusion of the particles (see Figure 1 for an enlarged side view). The crossflow * To whom correspondence should be addressed. Myeong Hee Moon, Department of Chemistry, Yonsei University, Seoul, 120-749, South Korea. Phone, 82 2 2123 5634; fax, 82 2 364 7050; e-mail, mhmoon@yonsei.ac.kr. † Yonsei University. ‡ Gachon University of Medicine and Science. 10.1021/pr800225z CCC: $40.75  2008 American Chemical Society Journal of Proteome Research 2008, 7, 3475–3480 3475 Published on Web 06/21/2008