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Supplementary Materials and Methods
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1.1. Lipid extraction and derivatization for PI, LPI analysis.Total 20μg EV phospholipids
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were extracted by Bligh & dyer method[36]. Briefly, EV were directly transferred into 3 ml of
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CHCl3: methanol (1:2, v/v) in 15ml conical glasstube and spikedphosphatidylcholine (PC)
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(10:0-10:0), phosphatidylglycerol (PG) (10:0-10:0), phosphatidylinositol (PI) (8:0-8:0),
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lysophosphatidylcholine
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sphingomyelin (SM) (d18:1-12:0). For PI, LPI analysis, a solution of TMSD (2 mol/L) in
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hexane (50 µL) was added to the lipid extracts dissolved in methanol to obtain
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yellow-colored solutions. After vortexing for 30 s, methylation was performed at 37°C for 20
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min. Addition of glacial acetic acid (6 µL) quenched the methylation and afforded colorless
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samples, which were then subjected to LC/MS.
(LPC)
(C13:0),
lysophosphatidylinositol
(LPI)
(C13:0),
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1.2. Quantification of target lipids in PC9, PC9R EV using Triple Quadrupole
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LC-MS.The quantification of target lipids in EV samples was performed by 6490
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Accurate-Mass Triple Quadrupole (QqQ) LC-MScoupled to a 1200 series HPLC system
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(Agilent Technologies, Wilmington, DE, USA) with a Hypersil GOLD column (2.1 × 100 mm
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ID; 1.9 μm, Thermo science).This provides high sensitivity byiFunnel technology that
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consists of three components: Agilent Jet Stream technology, a hexabore capillary, and a
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dual ion funnel. The flow rate was 0.1 mL/min and the injection volume was 5 μL for each
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run.The gradient elution program consisted of holding solvent steady (A/B: 95/5) for 10 min;
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followed by a first linear gradient to solvent (A/B: 70/30) for 20 min; followed by a second
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linear gradient to solvent (A/B: 10/90) for 30 min; and terminating with isocratic elution at
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solvent (A/B: 10/90) for 10 min. The column was equilibrated at 5% solvent B for 5 min
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before reuse. Total run time was 65 min for each analysis. All acquisition methods used the
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following parameters: 3500 V positive mode of capillary voltage, 3000 V negative mode of
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capillary voltage, a sheath gas flow of 11 L/min (UHP nitrogen) at 200ºC, a drying gas flow
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of 15 L/min at 150ºC, nebulizer gas flow at 25 psi. Multiple reaction monitoring (MRM)
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conditions including transition and MS/MS collision energy were optimized to analyze target
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lipids quite different in MALDI-TOF analysis.
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1.3 Lipids quantification and data analysisBy using an ODS column, various lipid
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species were separated according to their Cn and Un. For the compound with a higher Cn
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and a lower Un, the RT increased. For data analysis, each peak mustbe selected on the
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basis of a reasonable RT with high repeatability. Thus, we applied several internal
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standards to identify various lipid species.
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Supplementary Figure Legends
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Supplementary Table 1.Optimized MRM conditions of target lipids were shown.
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Figure S1. Comparison of MALDI-TOF MS spectra of phospholipids.Average positive ion
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MALDI-TOF mass spectra of the lipid extracts of PC9R_cell (blue), PC9_cell (red),
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PC9R_EV (green) and PC9_EV (yellow) was shown in bottom. The separated spectra of
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each PC9R_cell, PC9_cell, PC9R_EV and PC9_EV were shown in top.
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Figure S2. (A) A principal component analysis (PCA) plot for 30 PC9R_cell (blue), 30
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PC9_cell (red), 30 PC9R_EV (blue sky) and 30 PC9_EV (green). (B) Hierarchical clustering
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dendrograms of MS showing clustering patterns of PC9R_cell, PC9_cell, PC9R_EV and
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PC9_EV.
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Figure S3. (A) Heatmap of DRL obtained from positive ion mode with dendrogram,
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representing the up- (in red) or down-regulated (in green) phospholipid spectra in PC9_EV
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or PC9R_EV, compared to PC9R_EV or PC9_EV, respectively. (B) Heatmap of DRLs with
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dendrogram obtained in negative ion mode.
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Figure S4. Validation of identified DELs using MRM. The quantified peak areas were
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normalized by the spiked internal standard for each lipid class. The height of the bar and
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the error bar denote the average of the normalized peak area and the standard deviation of
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triplicate measurements, respectively(*p <0.1, **p < 0.05, Student’s t-test).
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Supplementary Table 1.
MS/MS CE
Lipids
Ion mode
MRM transitions
(eV)
PC
Positive
[M+H] > 184
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PG
Positive
[M+NH4] > [M+NH4–189]
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Methylated PI
Positive
[M+H] > [M+H–274]
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LPC
Positive
[M+H] > 184
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Methylated LPI
Positive
[M+H] > [M+H–274]
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SM
Positive
[M+H] > 184
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