High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells

Abstract

The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface.

Keywords: HPTLC-MS; exosomes; phospholipids; scanning densitometry.

Figure 1

(A) HPTLC chromatograms (UV 190 nm) of exosomes lipid extracts showing separation of phospholipid classes (SM, PC, PS, PE). B16-F1-EXOs: peak 1: SM (m.d. = 14.6 mm); peak 2: PC (m.d. = 20.3mm); peak 3: PS (m.d. = 23.8 mm) and peak 4: PE (m.d.=25.9 mm). B16-F10-EXOs: peak 1: SM (m.d. = 14.5 mm); peak 2: PC (m.d. = 18.0 mm); peak 3: PS (m.d. = 22.6 mm) and peak 4: PE (m.d.=25.6 mm) NIH-3T3-EXOs: peak 1: SM (m.d. = 14.0 mm); peak 2: PC (m.d. = 19.1 mm); peak 3: PS (m.d. = 21.4 mm) and peak 4: PE (m.d. = 24.8 mm). (B) Automatic extraction of HPTLC bands using the TLC-MS elution-based interface. Red: HPLC pump for MeOH delivery (0.2 mL/min); blue: ion trap MS equipment; black: frit for silicagel filtering; +: laser crosshair. Idealized operation of peaks 1 and 2 extraction: (a) bypass; (b) first band extraction; (c) air cleaning; (d) bypass; (e) second band extraction.

Figure 2

(A) HPTLC-ESI⁺-MS spectrum of SM standard. (B) HPTLC-ESI-MS/MS spectrum of the precursor ion at m/z 725.6 in the standard. (C) HPTLC-ESI⁺-MS spectrum of peak at 14.0 mm m.d. in NIH-3T3-EXOs sample. (D) HPTLC-ESI-MS/MS spectrum of the precursor ion at m/z 725.6, confirming peak to be SM in NIH-3T3-EXOs sample. (E) HPTLC-ESI⁺-MS spectrum of peak at 14.6 mm m.d. in B16-F1-EXOs sample. (F) HPTLC-ESI⁺-MS spectrum of peak at 14.5 mm m.d. in B16-F10-EXOs sample. For B16-F1 EXOs and B16-F10 EXOs, HPTLC-ESI-MS/MS spectra of the corresponding precursor ion at m/z 725.6 provides the same product ions than those of (B) and (D).

Figure 3

(A) HPTLC-ESI⁺-MS spectrum of PC standard. (B) HPTLC-ESI-MS/MS spectrum of the precursor ion at m/z 782.6 in the standard. (C) HPTLC-ESI⁺-MS spectrum of peak at 19.1 mm m.d. in NIH-3T3-EXOs sample. Most intense ion at m/z 768.7 (PC33:1). (D) HPTLC-ESI-MS/MS spectrum of the precursor ion at m/z 782.6 (PC34:1), confirming peak to be PC in NIH-3T3-EXOs sample. (E) HPTLC-ESI⁺-MS spectrum of peak at 20.3 mm m.d. in B16-F1-EXOs sample. (F) HPTLC-ESI⁺-MS spectrum of peak at 18.0 mm m.d. in B16-F10-EXOs sample.

Figure 4

Normalized profiles of lipid species in the corresponding SM and PC classes of studied exosomes, obtained by HPTLC-densitometry-ESI⁺-MS (X-axis: m/z of ions and their corresponding species.

Figure 4

Normalized profiles of lipid species in the corresponding SM and PC classes of studied exosomes, obtained by HPTLC-densitometry-ESI⁺-MS (X-axis: m/z of ions and their corresponding species.

Figure 5

(A) UV-190 (blue) and primuline-induced fluorescence (red) densitograms corresponding to B16F1-EXOs and B16F10-EXOs. (see Experimental for impregnation and Fluorescence densitometry conditions). (B) Results from one-plate quantification of SM and PC classes in exosome lipid extracts by UV at 190 nm and primuline-induced fluorescence densitometry, using response factors of the corresponding standards, and expressed in µg of PL per 100 µg of exosome protein.