| Summary: | 碩士 === 國立臺灣師範大學 === 化學系 === 104 === Sialylation plays important roles in many cellular functions. Due to the low abundance, low ionization efficiency of sialo-glycopeptides and frequently observed dissociation of sialic acid residues during enrichment processes, tools to enhance the enrichment specificity and detection sensitivity in LC-MS/MS are of great interest. Here, we apply ZIC-cHILIC that carries phosphorylcholine functional group where the positively charged amine group is exposed outside to be more accessible to enhance the electrostatic attraction of negatively charged carboxylic group of sialic acid to purify the intact glycopeptides. By using single step ZIC-cHILIC and Orbitrap Velos analysis, in total, 82, 76, 86, 97, 95 and 37 unique sialo-glycopeptide were identified among 117, 104, 108, 134, 136 and 52 unique glycopeptide in the 65%, 60%, 55%, 50%, 40 % ACN and 0% ACN /0.5% FA direct elution, respectively by using cHILIC. We further applied a stepwise elution strategy for fractionating sialoglycopeptides of different properties by different percentage of acetonitrile (ACN). Combining 5 fractions from 0-70% ACN elution through cHILIC fractionation, the number increase from 15 to a total of 32 intact sialo-glycopeptides (m/z from 3000 to 7500) from fetuin were observed in MALDI-TOF analysis. Furthermore, we applied this strategy to study the sialo-glycoproteome in non-small cell lung cancer cells (PC9 cells). On the proteome scale, we identified 751 and 1349 intact sialo-glycopeptides in PC9 NSCLC cells by MAGIC and Byonic software, respectively. Different glycan compositions located on bi-, tri- and tetra-antennary such as (1) antennary without any fucosylation or terminal sialylation; (2) antennary with core- or terminal fucosylation but without sialylation; and (3) antennary with terminal sialylation were identified in our strategy. Among these identified glycoproteins, 9 of 13 N-linked glycosylation sites of epidermal growth factor receptor (EGFR), one of important yet well-known marker in NSCLC cell, were identified by our strategy. In summary, using cHILIC stepwise fractionation demonstrated specificity to enrich intact sialo-glycopeptides and increase the coverage on proteome scale.
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