Multiple precursor ion scanning of gangliosides and sulfatides with a reversed-phase microfluidic chip and quadrupole time-of-flight mass spectrometry

Abstract

Precise profiling of polar lipids including gangliosides and sulfatides is a necessary step in understanding the diverse physiological role of these lipids. We have established an efficient method for the profiling of polar lipids using reversed-phase nano high-performance liquid chromatography microfluidic chip quadrupole time-of-flight mass spectrometry (nano-HPLC-chip Q-TOF/MS). A microfluidic chip design provides improved chromatographic performance, efficient separation, and stable nanospray while the advanced high-resolution mass spectrometer allowed for the identification of complex isobaric polar lipids such as NeuAc- and NeuGc-containing gangliosides. Lipid classes were identified based on the characteristic fragmentation product ions generated during data-dependent tandem mass spectrometry (MS/MS) experiments. Each class was monitored by a postprocessing precursor ion scan. Relatively simple quantitation and identification of intact ions was possible due to the reproducible retention times provided by the nano-HPLC chip. The method described in this paper was used to profile polar lipids from mouse brain, which was found to contain 17 gangliosides and 13 sulfatides. Types and linkages of the monosaccharides and their acetyl modifications were identified by low-energy collision-induced dissociation (CID) (40 V), and the type of sphingosine base was identified by higher energy CID (80 V). Accurate mass measurements and chromatography unveiled the degree of unsaturation and hydroxylation in the ceramide lipid tails.

Figure 1

(A) Base peak chromatograms (left panel) and mass spectra (right panel) of ganglioside standards GM3, GD3 and GT1b. A C18 analytical column was used as reverse-phase mode for the separation of ceramide moieties. 20-50 ng samples were injected. Inserts are the sugar structures of each ganglioside. Right panel mass spectra show gangliosides with ceramide d38:1 from LC separation, which is indicated by arrows on the left panel. Ganglioside subspecies and their assignments are shown in S-Table 1. (B) Retention time vs. m/z plot of the ganglioside standards. Ganglioside separation mainly depends on the hydrophobicity of ceramide moieties.

Figure 2

Separation of complex gangliosides on the nanoHPLC chip. (A) EIC of bovine milk ganglioside GD3 (d41:1) at m/z 769.952. Separation of the monoisotopic peak of NeuAc-NeuAc GD3 (d41:1) and the third isotopic peak of NeuAc-NeuAc GD3 (d41:2) was achieved. The contribution of ¹³C was removed by the LC. (B) EIC of NeuGc NeuAc GD3 (d40:1) at m/z 770.941. The separation of NeuAc containing gangliosides and NeuGc containing gangliosides was achieved. The first peak and second peak correspond to the monoisotope of NeuGc NeuAc GD3 (d40:1) and the third isotope of NeuAc-NeuAc GD3 (d41:1), respectively. All the ions are doubly depronated. The isotopic peaks of other compounds are indicated by asterisks (*). Inserts are the spectrum at each retention time.

Figure 3

Detection and identification of individual classes of polar lipids in mouse brain extracts. (A) A two dimensional plot of m/z vs. retention time for mouse brain polar lipids. (B) Base peak chromatogram (BPC) from MS analysis, (C) post processing precursor ion scan for m/z 290.095 (gangliosides), (D) for m/z 96.960 (sulfatide), and (E) for m/z 241.011 (phosphatidylinositol). The fragment ions for the individual headgroup being characteristic of their species can be used for identification of their lipids within one class, followed by alignment with the BPC. Gangliosides, which are the most polar lipids, eluted early in the chromatography. EICs with 10 ppm window were used for the scans. Both BPC and EICs were obtained in the negative ion mode. Details are described in the experimental section.

Figure 4

Low (40V) and higher (80V) collision energy MS/MS spectra of (A) ganglioside GT1b (d18:1/18:0) and (B) sulfatide (d18:1/16:0). Low energy spectra and high energy spectra contain various diagnostic ions for the headgroup and ceramide tails, respectively.