Glycomic Profiling of Tissue Sections by LC-MS

Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to online purification and LC-electrospray ionization (ESI)-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-μL aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak areas of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed

Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LC–MS/MS

Prostate specific antigen (PSA) is currently used as a diagnostic biomarker for prostate cancer. It is a glycoprotein possessing a single glycosylation site at N69. During our previous study of PSA N69 glycosylation, additional glycopeptides were observed in the PSA sample that were not previously reported and did not match glycopeptides of impure glycoproteins existing in the sample. This extra glycosylation site of PSA is associated with a mutation in KLK3 genes. Among single nucleotide polymorphisms (SNPs) of KLKs families, the rs61752561 in KLK3 genes is an unusual missense mutation resulting in the conversion of D102 to N in PSA amino acid sequence. Accordingly, a new N-linked glycosylation site is created with an N102MS motif. Here we report the first qualitative and quantitative glycoproteomic study of PSA N102 glycosylation site by LC–MS/MS. We successfully applied tandem MS to verify the amino acid sequence possessing N102 glycosylation site and associated glycoforms of PSA samples acquired from different suppliers. Among the three PSA samples, HexNAc2Hex5 was the predominant glycoform at N102, while Hex­NAc4­Hex5­Fuc1­Neu­Ac1 or Hex­NAc4­Hex5­Fuc1­Neu­Ac2 was the primary glycoforms at N69. D102 is the first amino acid of “kallikrein loop”, which is close to a zinc-binding site and catalytic triad. The different glycosylation of N102 relative to N69 might be influenced by the close vicinity of N102 to these functional sites and steric hindrance

N‑Linked Glycan Profiling in Neuroblastoma Cell Lines

Although <i>MYCN</i> amplification has been associated with aggressive neuroblastoma, the molecular mechanisms that differentiate low-risk, <i>MYCN</i>-nonamplified neuroblastoma from high-risk, <i>MYCN</i>-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 is important in cell signaling and immune response. Aberrant N-linked glycosylation has been routinely linked to various cancers. In particular, glycomic markers have often proven to be useful in distinguishing cancers from precancerous conditions. Here, we perform a systematic comparison of N-linked glycomic variation between <i>MYCN</i>-nonamplified SY5Y and <i>MYCN</i>-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 <i>MYCN</i>-amplified samples in comparison to smaller, nonsialylated glycans that are more dominant in the <i>MYCN</i>-nonamplified samples. These results offer clues for deriving marker candidates for accurate neuroblastoma risk diagnosis

LC–MS Profiling of N‑Glycans Derived from Human Serum Samples for Biomarker Discovery in Hepatocellular Carcinoma

Defining clinically relevant biomarkers for early stage hepatocellular carcinoma (HCC) in a high-risk population of cirrhotic patients has potentially far-reaching implications for disease management and patient health. Changes in glycan levels have been associated with the onset of numerous diseases including cancer. In the present study, we used liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI-MS) to analyze N-glycans in sera from 183 participants recruited in Egypt and the U.S. and identified candidate biomarkers that distinguish HCC cases from cirrhotic controls. N-Glycans were released from serum proteins and permethylated prior to the LC–ESI-MS analysis. Through two complementary LC–ESI-MS quantitation approaches, global profiling and targeted quantitation, we identified 11 N-glycans with statistically significant differences between HCC cases and cirrhotic controls. These glycans can further be categorized into four structurally related clusters, matching closely with the implications of important glycosyltransferases in cancer progression and metastasis. The results of this study illustrate the power of the integrative approach combining complementary LC–ESI-MS based quantitation approaches to investigate changes in N-glycan levels between HCC cases and patients with liver cirrhosis