Table 1.

The proteomic studies on melanoma-derived EVs (or secretome).

Source of EVs	Types of Isolated EVs	Methods of Isolation	Applied Proteomic Techniques	Number of Identified Proteins	Major Findings	Ref.
MeWo and SK-MEL-28 human melanoma cell lines	exosomes	0.1 µm filtration followed by ultracentrifugation at 100,000× g	2D SDS-PAGE-MS/MS (MALDI-TOF)	Forty-nine common protein spots in the exosome samples corresponding to 41 different proteins	p120 catenin, radixin, and immunoglobulin superfamily member 8 were identified in exosomes for the first time; mitochondrial and lysosomal proteins were significantly reduced in exosomes, confirming the endosomal origin of exosomes	[26]
MNT-1, G1 and Mel501 (non-tumorigenic), Daju and SK-MEL-28 (tumorigenic), A375 and 1205Lu (metastatic) human melanoma cell lines	exosomes	ultracentrifugation (100,000× g) and separation in sucrose (0.25–2.5 M) density gradient	1D SDS-PAGE followed by nano LC-MS/MS	between 486 and 632 depending on the cell line (517 on average); 917 unique proteins in all samples	exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response that were less-abundant or absent in exosomes from less-aggressive cells	[28]
Mel501 human melanoma cell line cultured in standard or acidic (pH 6.0) conditions	exosomes	ultracentrifugation (130,000× g) and separation in iodixanol (OptiPrep) gradient	1D SDS-PAGE followed by RPLC-MS/MS	three replicates for exosomes from pH 6.0: 212, 211, and 217 proteins; control: 194, 239, and 130 proteins	lower pH 6.0 modified exosome protein profile, causing up-regulation of more than 50% of the proteins	[30]
B16-F1 melanoma cell line (established from C57BL/6 mouse)	exosomes, ectosomes, apoptotic bodies	centrifugation at 25,000× g to pellet ectosomes and apoptotic bodies; the remaining supernatant was then filtered (0.22 μm) and exosomes were pelleted at 100,000× g; exosomes and apoptotic bodies were further purified by discontinuous sucrose cushion or linear sucrose gradient, respectively	uHPLC-MS (nanospray source of a LTQ Orbitrap XL)	553 proteins common to all populations	procoagulant proteins were more abundant in ectosomes and apoptotic bodies than in exosomes, with tissue factor (and lipid-phosphatidylserine) critical for procoagulant activity	[24]
B16F10 mouse melanoma cell line	exosomes, ectosomes	10,000× g to pellet ectosomes; exosomes were then pelleted from the remaining supernatant at 110,000× g; sucrose density and Nycodenz gradients were also applied to further separate exosomes; independent isolation of exosomes was performed using size exclusion chromatography (SEC)	nanoLC-MS/MS	a total of 4421 proteins; 1540 proteins common to all populations, and 533, 354, and 110 proteins were identified exclusively in ectosomes and low- and high-density exosomes, respectively	bottom-loading (instead of top-loading) of exosomes on sucrose density and Nycodenz gradients resulted in separation of high- and low-density exosomes displaying distinct protein profiles	[29]
melanoma A375 and normal melanocytic HEMa-LP cell lines	exosomes	500 kDa cut-off ultrafiltration followed by ultracentrifugation at 100,000× g, Exoquick-TC precipitation	2D DIGE-LC-MS/MS	total number not provided, 114 protein spots detected	differential expression of annexin A1, annexin A2, syntenin-1, and hyaluronian and proteoglycan link protein 1 between melanocytes and melanoma exosomes	[27]
primary cell cultures established from 14 original tumor specimens of uveal melanoma patients	not isolated, entire secretome was analyzed	-	label-free nanoLC-MS/MS	total of 1843 proteins (758 with at least 3 unique peptides)	a subsets of 83 up-regulated and 80 down-regulated proteins in uveal melanoma secretome	[38]
sera of uveal melanoma patients and healthy controls	not isolated, entire secretome was analyzed	-	2D SDS-PAGE- LS-MS (CapLC system coupled to a Q-TOF spectrometer)	133 (on average)	cathepsin D and gp100 protein levels increased in sera of uveal melanoma patients	[25]