LAUSR

Abstract Thin-layer chromatography (TLC) combined with infrared MALDI mass spectrometry (IR-MALDI-MS) is a powerful analytical tool for detection and structural characterization of glyco- and phospholipids directly from the TLC plate. Here we coupled a pulsed IR-MALDI laser to a hybrid Synapt G2-S mass spectrometer (Waters) and obtained an effective focal spot size of ∼50 × 70 μm2 in diameter. We used this new MALDI ion source configuration for TLC-IR-MALDI-MS imaging of neutral glycosphingolipids (GSLs), obtained from human colon epithelial HCT-8 cells, at 100 μm step size. Our analytical focus was on the detection of globo-series GSLs globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the main receptors for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC). The direct TLC-IR-MALDI-MSI analysis allowed precise visualization of the chromatographic separation of the various lipoforms of Gb3Cer and Gb4Cer, with ceramide moieties mainly ranging from Cer (d18:1, C16:0) to Cer (d18:1, C24:0/C24:1) as well as those of their precursor GSLs glucosylceramide (GlcCer) and lactosylceramide (Lc2Cer). Reference TLC overlay immunostaining assays were conducted on parallel developed TLC lanes for confirmation of anomeric sugar configuration of proposed structures. Together, the adopted protocol provided a rapid and near-comprehensive overview of the GSL composition of the investigated cell line of high medical relevance. This possibility could also be highly useful in glycolipidomics studies of complex biological matrices.