Sola spe plates

To extract the exosomal proteins, exosomes were lysed by RIPA buffer (89900, Thermo Fisher Scientific; Waltham, MA). The proteins were then digested with trypsin (60109-101, SMART Digest Trypsin Kit, Thermo Fisher Scientific; Waltham, MA), desalted (Z720070, Millipore Ziptips Micro-C18; Sigma-Aldrich, Milwaukee, WI), purified (60309-001, SOLA™ SPE Plates; Thermo Fisher Scientific; Waltham, MA), and dissolved in 0.1% formic acid for LC-MS/MS analysis (LTQ Orbitrap Velos, Thermo Scientifics; Waltham, MA) (service provided by the Mass Core Facility of Genomics Research Center, Academia Sinica). The acquired proteomics raw data files were then searched against a UniProt human protein database (http://www.uniprot.org/) by using PEAKS Studio 7.5 (PEAKS Studio, Bioinformatics Solutions, Waterloo, Ontario, Canada). The following settings were used in PEAKS Studio 7.5 in conjunction with UniProt to search the protein database: enzyme set to trypsin with a maximum of two missed cleavage site precursor and fragment mass tolerance of 20 ppm and 0.8 Da, respectively. Finally, the spectral counts obtained from each peptide were normalized to the total spectral counts recorded for all peptides in a sample.

Ganglioside classes GM1, GD1, GD2, GD3, GT1, GT2, GT3, and GQ1 were extracted and analyzed as follows. Gangliosides in the remaining water phase of the two-step chloroform:methanol procedure were subjected to purification using solid-phase extraction (Thermo Scientific SOLA SPE plates, 10 mg/2 mL)^{47 (link)}. The water phase was loaded on columns pre-washed with chloroform:methanol (2:1, V:V), methanol and methanol:water (1:1, V:V); with the input flow through re-applied three times. Then, columns were washed with water, and the elution was carried out two times with methanol and one time with chloroform:methanol (1:1, V:V). Washing and elution steps were carried using a vacuum manifold. Pooled eluates were dried in a speed vacuum concentrator and re-suspended in 33% ethanol solution of methylamine in chloroform:methanol (0.003:5:1; V:V:V). Ganglioside extracts were analyzed by direct infusion on a QExactive MS (Thermo Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in negative ion modes with a resolution of Rm/z = 200 = 140,000; AGC target of 1e6; maximum injection time of 500 ms and 3 microscans.