Comprehensive lipidome profiling of isogenic primary and metastatic colon adenocarcinoma cell lines

Abstract

A "shotgun" lipidomics strategy consisting of sequential functional group selective chemical modification reactions coupled with high-resolution/accurate mass spectrometry and "targeted" tandem mass spectrometry (MS/MS) analysis has been developed and applied toward the comprehensive identification, characterization and quantitative analysis of changes in relative abundances of >600 individual glycerophospholipid, glycerolipid, sphingolipid and sterol lipids between a primary colorectal cancer (CRC) cell line, SW480, and its isogenic lymph node metastasized derivative, SW620. Selective chemical derivatization of glycerophosphoethanolamine and glycerophosphoserine lipids using a "fixed charge" sulfonium ion containing, d(6)-S,S'-dimethylthiobutanoylhydroxysuccinimide ester (d(6)-DMBNHS) reagent was used to eliminate the possibility of isobaric mass overlap of these species with the precursor ions of all other lipids in the crude extracts, thereby enabling their unambiguous assignment, while subsequent selective mild acid hydrolysis of plasmenyl (vinyl-ether) containing lipids using formic acid enabled these species to be readily differentiated from isobaric mass plasmanyl (alkyl-ether) containing lipids. Using this approach, statistically significant differences in the abundances of numerous lipid species previously identified as being associated with cancer progression or that play known roles as mediators in a range of physiological and pathological processes were observed between the SW480 and SW620 cells. Most notably, these included increased plasmanylcholine and triglyceride lipid levels, decreased plasmenylethanolamine lipids, decreased C-16 containing sphingomyelin and ceramide lipid levels, and a dramatic increase in the abundances of total cholesterol ester and triglyceride lipids in the SW620 cells compared to those in the SW480 cells.

Figure 1

Positive ion mode ESI high-resolution mass spectrometric analysis of crude lipid extracts from the human adenocarcinoma cell lines (A) SW480 and (B) SW620. An internal standard lipid PC_(14:0/14:0) was included to enable relative quantification of the observed lipid species, while the internal standard lipids PE_(14:0/14:0) and PS_(14:0/14:0) (not labeled) were included to allow completion of subsequent reaction with d₆-DMBNHS to be monitored.

Figure 2

Positive ion mode ESI high-resolution mass spectrometric analysis of a crude lipid extract from the SW480 human adenocarcinoma cell line, obtained (A) after reaction with the amine-specific derivatization reagent, d₆-DMBNHS, and (B) after reaction with d₆-DMBNHS followed by mild formic acid hydrolysis.

Figure 3

Quantification of PC lipids from the d₆-DMBNHS derivatized SW480 and SW620 cell crude lipid extracts. (A) Percent individual PC ion abundances compared to the total ion abundance for all PC lipids. (B) Percent total ether-linked PC ion abundance compared to the total PC lipid ion abundance. (C) Percent total PC ion abundance compared to the total ion abundance for all identified lipids. n=5, *=p<0.01.

Figure 4

Quantification of PE lipids from the d₆-DMBNHS derivatized SW480 and SW620 cell crude lipid extracts. (A) Percent individual PE ion abundances compared to the total ion abundance for all PE lipids. (B) Percent total ether-linked PE ion abundance compared to the total PE lipid ion abundance. (C) Percent total PE ion abundance compared to the total ion abundance for all identified lipids. n=5, *=p<0.01.

Figure 5

Quantification of TG lipids from the d₆-DMBNHS derivatized SW480 and SW620 cell crude lipid extracts. (A) Percent individual TG ion abundances compared to the total ion abundance for all TG lipids. (B) Percent total ether-linked TG ion abundance compared to the total TG lipid ion abundance. (C) Percent total TG ion abundance compared to the total ion abundance for all identified lipids. n=5, *=p<0.01.