Wide-Targeted Semi-Quantitative Analysis of Acidic Glycosphingolipids in Cell Lines and Urine to Develop Potential Screening Biomarkers for Renal Cell Carcinoma

Abstract

Glycosphingolipids (GSLs), mainly located in the cell membrane, play various roles in cancer cell function. GSLs have potential as renal cell carcinoma (RCC) biomarkers; however, their analysis in body fluids is challenging because of the complexity of numerous glycans and ceramides. Therefore, we applied wide-targeted lipidomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM) based on theoretical mass to perform a comprehensive measurement of GSLs and evaluate their potency as urinary biomarkers. In semi-quantitative lipidomics, 240 SRM transitions were set based on the reported/speculated structures. We verified the feasibility of measuring GSLs in cells and medium and found that disialosyl globopentaosylceramide (DSGb5 (d18:1/16:0)) increased GSL in the ACHN medium. LC-MS/MS analysis of urine samples from clear cell RCC (ccRCC) patients and healthy controls showed a significant increase in the peak intensity of urinary DSGb5 (d18:1/16:0) in the ccRCC group compared with that in the control group. Receiver operating characteristic analysis indicated that urinary DSGb5 could serve as a sensitive and specific marker for RCC screening, with an AUC of 0.89. This study demonstrated the possibility of urinary screening using DSGb5 (d18:1/16:0). In conclusion, urinary DSGb5 (d18:1/16:0) was a potential biomarker for cancer screening, which could contribute to the treatment of RCC patients.

Keywords: disialosyl globopentaosylceramide (DSGb5); glycosphingolipids; liquid chromatography/tandem mass spectrometry (LC–MS/MS); renal cell carcinoma; targeted lipidomics.

Figure 1

Representative glycosphingolipid structure. This structure shows disialosyl globopentaosylceramide (DSGb5).

Figure 2

Symbol Nomenclature for Glycans (SNFG): Cer, ceramide. This symbol shows the DSGb5.

Figure 3

Biosynthetic pathway of GSLs: MSGb5, monosialosyl globopentaosylceramide; DSGb5, disialosyl globopentaosylceramide; GM3, monosialodihexosylganglioside; GM2, monosialotrihexosylganglioside; GM1, monosialotetrahexosylganglioside; DUPAN-2, Duke pancreatic monoclonal antigen type 2; GD1a, ganglioside disialic acid 1a; DSLc4, α(2,3)/α(2,6) disialyl lactotetraosylceramide; GalNAcDSLc4, N-acetylgalactosaminyl disialyl lactotetraosyl ceramide; CA19-9, carbohydrate antigen 19-9. The symbols are notated in the same way as in Figure 2.

Figure 4

In silico predicted SRM conditions for wide-targeted GSL analysis. For SRM analysis, 240 transitions were set for GSLs consisting of 10 glycans and 24 FA chains. The m/z of Q1 was calculated from the combination of ceramide and glycans, and Q3 was set at m/z 290, derived from sialic acid: CID, collision-induced dissociation; FA, fatty acid; SRM, selected reaction monitoring.

Figure 5

Representative product ion spectrum of GD1a (d18:1/18:0). m/z 918 is [M-2H]²⁻ of GD1a (d18:1/18:0). m/z 290, which is derived from NeuAc moiety, was detected as the most intense product ion: GD1a, ganglioside disialic acid 1a.

Figure 6

Typical SRM chromatograms for wide-targeted GSL analysis: (a) HK-2 cells, (b) ACHN cells, (c) HK-2 medium, and (d) ACHN medium. Each colored SRM chromatogram corresponds to a theoretical GSL structure: SRM, selected reaction monitoring.

Figure 7

Screening performance of urinary DSGb5 levels (d18:1/16:0): (a) DSGb5 (d18:1/16:0) peak intensities in the urine of healthy controls and RCC patients; (b) ROC analysis and the performance status of urinary DSGb5 levels (d18:1/16:0). The urinary DSGb5 (d18:1/16:0) peak intensity was significantly higher in RCC patients than that in healthy controls. The screening performance of DSGb5 (d18:1/16:0) was very high.