Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane, which play significant roles in organism. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a new method to analyze GSL glycans by a lectin microarray.

Glycosphingolipids (GSLs) are essential components of mammalian membranes and control many cellular processes such as cell adhesion, apoptosis, proliferation, intracellular homeostasis, angiogenesis, inflammatory response, membrane structure and subsequently cell signaling [1,2]. Two building blocks of GSLs are glycan structures extending into the extracellular matrix and a lipid moiety anchoring the outer leaflet of cell membranes, which makes GSL an amphiphilic molecule.

Using various glycosphingolipidomics analysis techniques such as high-performance liquid chromatography and mass spectrometry, researchers could increase understanding of GSL-related diseases. However, due to the complexities of the glycan head group as well as the ceramide chain-length heterogeneity, challenges focus on both structural and functional research in the field of glycosphingolipidomics are still worth noting [3,4]. For example, a liquid chromatography separation is imperative before the MS analysis, and it is difficult, time consuming and expensive to distinguish each GSL class and to obtain precise glycan profiles of GSLs [5,6].

In the past years, after the advent of the lectin microarray technology, researchers could use a lectin microarray to analyze glycopatterns of glycoproteins from multiple clinical samples of different diseases such as serum [7,8], tissues [9], urine [10,11], and saliva [12,13], demonstrating valid observation in terms of biomarker studies and, as a result, proofing that the lectin microarray is an effective and reliable tool in the field of glycomics, which laid the foundation for this protocol being reported.

Our group has recently developed an integrated strategy to analyze GSL glycans from different cell lines using lectin microarrays and MALDI-TOF mass spectrometry [6]. Here we describe how lectin microarrays and MS can be used to analyze GSL glycans. This protocol consists of extraction of GSLs from cell pellets, N-monodeacylation by using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, fluorescence labeling at the newly exposed amine group, preparation of lectin microarrays, GSL glycan analysis by lectin microarrays, complementary MS analysis, and data acquisition and analysis. A comprehensive annotation based on the results of the lectin microarrays and MALDI-TOF MS can present the real glycan structures containing different linkages of GSL glycans.


Figure 1. Flowchart of the comprehensive analysis of GSL glycans by lectin microarrays and MALDI-TOF MS from liver cell lines [6]. (A) Workflow of lectin microarray analysis of GSL glycans. (B) Workflow of detection of GSL glycans using MALDI-TOFmass spectrometry.

Using lectin microarrays to analyze GSL glycans from cultured cells directly detects and reveals glycopatterns of GSL extracts without the need for glycan release and allows the simultaneous large-scale profiling and monitoring of GSL glycans. Combined with supplementary data from MALDI-TOF/TOF-MS analysis, application of this strategy enabled the identification of exact structures of GSL glycans from various biological samples. This should increase the growing understanding for many cellular processes and enable new insight into GSL-related diseases. We present illustrative results here from one normal human liver cell line and three human hepatocarcinoma cell lines with different metastasis potentials. We anticipate that the procedure could be followed on different cell lines; however, the optimal number of cells to harvest might need to be adapted depending on cell type and cell culturing method.

This is the first time that the analysis of GSL glycans using a lectin microarray to be reported. By developing this new method, we hope that we could provide a higher throughput and easier approach, and decipher the so-far-enigmatic GSL language.



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