Escape from NK cell tumor surveillance by NGFR-induced lipid remodeling in melanoma

Metabolomics Data used in the publication

1) raw files obtained by the FGCZ (Functional Genomics Center Zurich):

QCpools (technical replicate of all sample pooled: p2947_o5292_SOP3_DDA5_pos_QCpool_cells

Sample of M010817 CMVTOEV cells not induced: p2947_o5292_SOP3_DDA5_pos_EV_cells_noninduced_sample

Sample of M010817 CMVTOEV cells induced: p2947_o5292_SOP3_DDA5_pos_EV_cells_induced_sample

Sample of M010817 CMVTONGFR cells not induced: p2947_o5292_SOP3_DDA5_pos_p75_cells_noninduced_sample

Sample of M010817 CMVTONGFR cells induced: p2947_o5292_SOP3_DDA5_pos_p75_cells_induced_sample

2) method description LC-MS

LC-MS-based lipidomic analysis was performed with M010817 CMVTOEV and CMVTONGFR cells pretreated with 1 μg/ml doxycycline for 24 h. Cells were detached with PBS 2 mM EDTA, washed and resuspended in 1-butanol/methanol (1:1). Cells were vortexed for 15 sec and subsequently sonicated on ice with a pulse of 3x 10 sec and 1x 30 sec at an amplitude of 15% using a SONOPULS HD 2070 Ultrasonic Homogenizer (Bandelin). Cell extracts were centrifuged at 16’000 g, 20°C for 10 min to remove macromolecules and subsequently diluted (1:3) with water/methanol (1:2) solution. The dilution was vortexed and centrifuged (16,000 x g, 20 °C, 10 min). 100 μl of the supernatant was transferred to a glass vial with narrowed bottom (Total Recovery Vials, Waters) for LC-MS injection. 
Lipids were separated on a nanoAcquity UPLC (Waters) equipped with a HSS T3 capillary column (150 μm x30mm, 1.8 μm particle size, Waters), applying a gradient of 5 mM ammonium acetate in water/acetonitrile 95:5 (A) and 5 mM ammonium acetate in isopropanol/acetonitrile 90:10 (B) from 5% B to 100% B over 10 min. The following 5 min conditions were kept at 100% B, followed by 5 min reequilibration to 5% B. The injection volume was 1 μL. The flow rate was constant at 2.5 μl/min. The UPLC was coupled to QExactive mass spectrometer (Thermo) by a nanoESI source. MS data was acquired using positive polarization and data-dependent acquisition (DDA). Full scan MS spectra were acquired in profile mode from 80-1200 m/z with an automatic gain control target of 1e6, an Orbitrap resolution of 70`000, and a maximum injection time of 200 ms. The 5 most intense charged (z = +1 or +2) precursor ions from each full scan were selected for collision induced dissociation fragmentation. Precursor was accumulated with an isolation window of 0.4 Da, an automatic gain control value of 5e4, a resolution of 17`500, a maximum injection time of 50 ms and fragmented with a normalized collision energy of 20, 30 and 40 (arbitrary unit). Generated fragment ions were scanned in the linear trap. Minimal signal intensity for MS2 selection was set to 500.

3) csv result files were generated by the FGCZ (Functional Genomics Center Zurich)

adducts found: adducts_p2947_o5292_new_10K_20210910

identifications found: identifications_p2947_o5292_new_10K_20210910

abundances of features: measurements_p2947_o5292_new_10K_20210910

Metaboanalyst file statistics: Metabo_p2947_o5292_10k_cells_CVcleaned_20210916

Metaboanalyst file enrichment: Metabo_Enrichment_p2947_o5292_10k_cells_20210910 _p75

4)  method description data analysis

Data sets were evaluated with Progenesis QI software (Nonlinear Dynamics), which aligns the ion intensity maps based on a reference data set, followed by a peak picking on an aggregated ion intensity map. Detected ions were identified based on accurate mass, detected adduct patterns and isotope patterns by comparing with entries in the LipidMaps Data Base (LM) and KEGG database. Considered adducts were M+H, M+NH4, 2M+H M+H-H2O. A mass accuracy tolerance of 5 ppm was set for the searches. Fragmentation patterns were considered for 600 the identifications of metabolites. Putative identifications were further ranked based on Mass error (observed mass – exact mass), isotope similarity (observed versus theoretical).