N-Linked Glycan Profiling in Neuroblastoma Cell Lines

Abstract

Although MYCN amplification has been associated with aggressive neuroblastoma, the molecular mechanisms that differentiate low-risk, MYCN-non amplified neuroblastoma from high-risk, MYCN-amplified disease are largely unknown. Genomic and proteomic studies have been limited in discerning differences in signaling pathways that account for this heterogeneity. N-linked glycosylation is a common protein modification resulting from the attachment of sugars to protein residues and important in cell signaling and immune response. Aberrant N-linked glycosylation has been routinely linked to various cancers. In particular, glycomic markers have often proved useful in distinguishing cancers from precancerous conditions. Here, we perform a systematic comparison of N-linked glycomic variation between MYCN-non-amplified SY5Y and MYCN-amplified NLF cell lines with the aim of identifying changes in sugar abundance linked to high-risk neuroblastoma. Through a combination of liquid chromatography-mass spectrometry and bioinformatics analysis, we identified 16 glycans that show a statistically significant change in abundance between NLF and SY5Y samples. Closer examination revealed the preference for larger (in terms of total monosaccharide count) and more sialylated glycan structures in the MYCN-amplified samples in comparison to smaller, non-sialylated glycans that are more dominant in the MYCN-non-amplified samples. These results offer clues for deriving marker candidates for accurate neuroblastoma risk diagnosis.

N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues

“…Similarly, changes in the expression of long chains of lactosamine (polyLacNAc) have also been reported to be associated with cancer (15). Likewise, terminal modifications of glycans on the cancer cell surface, such as fucosylation, give rise to the presence of Lewis and sialyl Lewis antigens that have been implicated in tumor progression and metastasis (22)(23)(24)(25)(26). These aberrant glycosylations play a significant role in malignant transformation and therefore afford a valuable opportunity to exploit cancer-specific protein glycosylation markers for prognosis and diagnosis (16).…”

“…Mass spectrometry (MS)-based glycomic methodologies are now regularly used for the reliable profiling of glycans from clinical samples (17)(18)(19)(20)(21)(22). In fact, MS identification and measurement of glycans are being pursued as a structurallyinformative approach as opposed to the clinically-established immunological assays (23)(24)(25) or arrays (26).…”

Characterization of isomeric glycan structures by LC‐MS/MS

“…Glycomics changes associated with trumatic brain injury were also assessed using the above LC‐MS/MS analysis of permethylated glycans . Glycans derived from glioma stem cell xenografts , neuroblastoma and breast cancer cell lines, IgG , prostate‐specific antigen , and human milk, bovine milk, and goat milk were also analyzed using RPLC‐MS/MS of reduced and permethylated glycans.…”

“…Sensitive quantitative glycomics for low abundance structures in complex biological samples is still challenging . The reliability and sensitivity of glycan analysis can be improved by SRM or multiple‐reaction monitoring (MRM) using a triple quadrupole mass spectrometer . SRM or MRM quantitation is based on using transition ions that are observed in the tandem mass spectrum of the analyte of interest, not full mass scans.…”

“…MRM was first used for the analysis of glucose tetra‐oligosaccharides (Glcα1‐ 6Glcα1‐4Glcα1‐4Glc) in urine, urine spots, and plasma . Thus far, MRM has been applied to the analysis of reduced native glycans , 1‐phenyl‐3‐methyl‐5‐pyrazolone labeled glycans , and permethylated glycans . We recently applied the RPLC method described above in conjunction with MRM to sensitively quantify glycans derived from biological samples such as human blood serum .…”