以兩性離子親水層析法剖析唾液酸醣蛋白體

唾液酸化在許多細胞的功能表現中扮演一個很重要的角色。由於低含量、唾液酸醣胜肽離子低游離效率以及在純化的過程中，常常會發現唾液酸會脫離醣胜肽，因此用來提升純化唾液酸醣蛋白專一性以及偵測極限的分析工具成為了學者們感興趣的題目。在這邊，我們使用了正電荷基團裸露在材料外側的兩性離子親水層析管柱(ZIC-cHILIC)來純化唾液酸醣胜肽，希望能夠提升正電荷基團與帶負電荷的唾液酸之間的電子交互作用力進而提升純化的效率。藉由使用兩性親水離子層析管柱搭配直接沖堤分析物的方法並且利用質譜儀進行偵測，我們分別使用65%、60%、55%、50%和40 % ACN 進行直接冲堤，鑑定到了117、104、108、 134、136和52條醣胜肽，其中含有82、76、86、97、95和37條唾液酸醣胜肽。另外，我們也進一步採取階段式冲堤分析物的方法，使用不同百分比的乙腈冲堤出不同梯度的分析物。我們透過了兩性親水離子層析管柱並且利用0%-70%的乙腈冲堤並結合五個梯度的分析物，可以將使用基質輔助雷色脫附游離飛行質譜儀偵測到的唾液酸醣胜個數共32條(質荷比區間在3000到7500)。除此之外，我們使用這個策略對非小型細胞肺癌細胞株之中的唾液酸醣蛋白體進行分析。在蛋白質體學的分析中，我們分別使用了MAGIC和Byonic軟體，分別鑑定到了751條、1349條唾液酸醣胜肽。不同類型的醣結構，分別由兩個、三個、四個分支組成，像是 (1) 分支中沒有岩藻醣或是末端有唾液酸醣修飾(2) 分支中有岩藻醣或是末端有唾液酸醣修飾(3) 分支中僅有末端唾液酸醣修飾。並且，在這些醣胜肽中包含了在非小型細胞肺癌細胞株中十分重要的蛋白質EGFR蛋白13個醣基化點中的9個。總括來說，我們使用了兩性離子親水層析管柱並且搭配階段式梯度冲堤法有效的提升了鑑定唾液酸醣胜肽的專一性，並且增加了蛋白質體的覆蓋率。

關鍵字

兩性離子親水層析法；唾液酸醣胜肽；醣修飾；質譜儀

並列摘要

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.

並列關鍵字

cHILIC ； sialo-glycopeptides ； glycosylation ； mass spectrometry

參考文獻

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