Direct analysis of sialylated or sulfated glycosphingolipids and other polar and neutral lipids using TLC-MS interfaces

Abstract

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids.

Keywords: collision-induced dissociation; gangliosides; phospholipids; sphingomyelins; sulfatides; tandem mass spectrometry; thin-layer chromatography.

Fig. 1.

Structures and negative-ion fragmentation assignments of polar lipid standards used in this study. A: A ST. B: The ganglioside GD1a. Each sample contains structural variants. The STs and disialogangliosides are detected as singly charged negative ions [M – H]⁻ and doubly charged negative ions [M – 2H]²⁻, respectively. The fragmentation pattern for the d18:1/C18:0 GD1a homolog is shown in (B).

Fig. 2.

Sensitivity tests for the LESA TLC-MS method (negative-ion mode) as a function of the amount of GD1a (A) and d18:1/C24:1 ST (B) spotted on the TLC plate prior to the separation.

Fig. 3.

Evaluation of different spots on the same GD1a band by LESA TLC-MS (negative ion mode) (left side). Determination of the efficiency of reextraction in LESA TLC-MS (right side). GD1a was applied onto a TLC plate and the plate was developed. The same GD1a spot was analyzed four times using LESA TLC-MS in the negative-ion mode.

Fig. 4.

Comparison of negative-ion mode spectra of a ganglioside obtained with the TLC-MS method coupled with LESA (A, B) and CAMAG TLC-MS interfaces (C, D). GD1a was deposited on the TLC plate prior to the separation. A, C: Negative ion mass spectra of GD1a shown in the region of [M – 2H]²⁻. B, D: CID MS/MS spectra of [M – 2H]²⁻ m/z 931.50. The insert in (B) shows the full m/z range but expands the signal intensity range 0–0.3. The labeled fragment ions have m/z values of 290.10 (B_1α, B_1β), 470.14 (C_2α), 592.57 (Y₀), 673.25 (C_3α), 754.63 (Y₁), 916.69 (Y_2α/Y_2β), 1,119.77 (Y_3α/Y_2β), 1,281.8 (Y_4α/Y_2β), 1,410.79 (Y_3α), and 1,572.9 (Y_4α or Y_2β), respectively.

Fig. 5.

Comparison of negative-ion mode spectra of a ST obtained with the TLC-MS method coupled with LESA (A, B) and CAMAG TLC-MS (C, D) interfaces. The ST was deposited on the TLC plate prior to the separation. A, C: Negative ion mass spectra of d18:1/C24:1 ST shown in the region m/z 560–960. B, D: CID MS/MS spectra of [M – H]⁻ m/z 888.62. Product ion assignments are given in Fig. 1.

Fig. 6.

Analysis of the bovine brain lipid extract in the negative ion mode of LESA and CAMAG TLC-MS interfaces coupled with ESI-MS/MS. TLC plates stained with orcinol (A) and primuline (C). Lane 1, monosialoganglioside mixture (GM1, GM2, and GM3); lane 2, disialoganglioside mixture (GD1a, GD1b, and GD3); lane 3, GT1b and GQ1b; lane 5, ST mixture (d18:1/C12:0 ST, d18:1/C24:1 ST, d18:1/C24:0 ST, and d18:1/C12:0 di-ST); and lanes 4 and 6, bovine brain total lipid extracts. B: The band containing each of the GD1 isomers was desorbed from the TLC plate by LESA; the [M – H]⁻ peaks of the two homologs were isolated in Q1 and subjected to CID MS/MS. *The signature fragments that differentiate the glycan structures of GD1a and GD1b.