A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells

Abstract

We describe a relatively simple, detergent-free, enzymatic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural analysis afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the analysis of tumour and cell-derived GSL-glycans. The method uses the potential of MS(2) fragmentation in negative ion ESI mode to characterize, in detail, the biologically relevant glycan structures derived from GSLs.

Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells

“…Whilst we were able to confirm the absence of nLc4 in the ∆ B3GNT5 cell line, the presence of the P 1 epitope was not readily detected in both the IGROV1 and ∆ B3GNT5 cells analyzed in this study. This could be due to the low expression of the P 1 epitope, coupled with the use of different MS instrumentation in this study (as compared to our previous study using PGC-LC-ESI-IT-MS/MS that was performed on P 1 – enriched cell populations of IGROV113). Moreover, this study used a larger PGC column inner diameter on a conventional LC platform performed which resulted in less sensitivity as opposed to the microflow LC platform performed previously using PGC-ESI-IT-MS/MS.…”

“…As part of our initiative to comprehensively characterize nsGSLs, we have recently reported the presence of paragloboside (nLc4, precursor of P 1 ) and P 1 pentasaccharide in tumor specimens and immortal ovarian cancer cells using two complementary methods; PGC-LC-ESI-MS/MS and flow cytometry131415. In this study, we extended the profiling of nsGSLs into three distinct groups; Normal (HOSE17-1, FT33-Tag, FT190 and FT237 which were suggested as a potential origin of epithelial ovarian cancer1617), Ovarian (IGROV1, SKOV3, BG1, and CAOV3), and Non-ovarian cancer cell lines (Ls174T, HeLa, HCT15, and HCT116).…”

“…Glycans were enzymatically released from extracted GSLs of parental and ∆ B3GNT5 IGROV1 cells and analyzed using negative mode UHPLC-ESI-QTOF-MS/MS. The assignment of glycan structures was facilitated using diagnostic MS2 fragment ions previously described for the analysis of N -glycans released from glycoproteins as well as GSL-derived glycans using negative mode LC-ESI-MS/MS1319.…”

“…We have previously shown in two separate studies, respectively, that ovarian cancer cells, IGROV1 and SKOV3, have higher levels of glycoprotein α2-6 sialylation as compared to non-cancer ovarian epithelial cells19 and nLc4 and P 1 are both expressed on IGROV1 and ovarian cancer tissue-derived GSL-glycans13. Hence, the reduction of α2-6 sialylation as a direct consequence of the B3GNT5 deletion in ovarian cancer cells is indicative of their complex associations within the glycan-processing pathway.…”

“…We recently reported the presence of the P 1 glycan in tissue samples14 as well as on P 1 -enriched IGROV1 ovarian cancer cell lines13, serving as a cell-recognition molecule through its binding to naturally occurring anti-glycan antibodies1429. The presence of nLc4 and P 1 was detected on IGROV1 cells using flow cytometry, in which P 1 was detected using P3NIL100 antibody, a well-validated monoclonal IgM antibody shown to specifically recognize the chemically synthesized P 1 trisaccharide (Galα1-4Galβ1-4GlcNAcβ-), as indicated by glycan array profiling performed in this study.…”