利用質譜技術分析百慕達花粉過敏原BG60的後修飾基位置

Abstract

蛋白質BG60是百慕達花粉中的一個重要過敏原，會導致氣喘，花粉熱和過敏性鼻炎等過敏病徵。根據先前對該蛋白質的相關研究，BG60蛋白具有醣化，且醣鏈可能對其致敏特性占有重要影響。BG60蛋白的醣鏈結構已被解出，大部份屬於 paucimannosidic type。特別的是，BG60蛋白其中一段胜肽片段和黃素結合位置的保守序列具有相似性，此胜肽含有24個胺基酸並由N端蛋白質定序法所決定。而分析BG60蛋白的光譜性質亦暗示此蛋白質屬於一種黃素蛋白。總和以上，本論文的目標著重於分析BG60蛋白的各種修飾基的位置，包括醣化和黃素的結合位置。 為了分析BG60蛋白的醣化位置，先利用MALDI-TOF質譜比較去醣化前後的BG60胜肽混合物的質譜圖差異。並進一步以MALDI-TOF/TOF質譜確定所有去醣化胜肽的序列。我們鑑定出BG60蛋白具有四個醣化位置：Asn88、Asn325、Asn354、Asn477。接著進一步將每段醣化胜肽以逆相高效能層析法分離出來，以了解每個醣化位置的醣鏈型態的差異。我們的結果提供一個較深入的資料以了解醣蛋白的微異質性 (microheterogeneity)。 在分析黃素結合位置方面，先將BG60蛋白以限制性酵素水解反應切出數個分子量較大的蛋白質切割片段，並用SDS-PAGE電泳分離後，收集具有螢光性質的片段則進行in-gel tryptic digestion。進一步將含有螢光性質的胜肽以逆相高效能層析法分離出來。N端蛋白質定序結果暗示螢光物質可能接在BG60蛋白的His113位置上。但是利用質譜分析的結果並無法證明此螢光物質確實為黃素分子。目前推測是一種黃素分子的衍生物。 藉由酵素水解反應、逆相高效能層析分離法和質譜分析技術，我們鑑定了BG60蛋白的四個醣化以及一個未知結構螢光分子的結合位置。此策略提供一種高效率和實用的工具來分析蛋白質的修飾基位置。

BG60 is an important allergen of Bermuda grass (Cynodon dactylon) pollen, which causes allergic diseases such as asthma, hey fever and allergic rhinitis. According to previous reports, BG60 has been identified as an N-linked glycoprotein and suggested that its carbohydrate moiety may be relevant to allergic diseases. Furthermore, the major structures of carbohydrate moiety were characterized as a paucimannosidic-type. In particular, a 24-residue internal peptide sequence was determined by N-terminal protein sequencing, which showed similarity to a conserved flavin-binding motif. Spectroscopic analysis also indicated BG60 was a flavoprotein. The aim of this study is to analyze these post-translational modification sites of BG60 including glycosylation and flavin-binding site. The analysis of N-linked glycosylation sites was performed by comparing the profile of MALDI-TOF MS spectra of protease-digested (trypsin and lys-C, respectively) peptide mixture with and without PNGase A treatment. The sequence of deglycosylated peptides were confirmed by MALDI MS/MS analysis. Four N-linked glycosylation sites were identified in BG60 : Asn88, Asn325, Asn354, Asn477. Furthermore, the four N-linked glycopeptides were purified by reverse-phase HPLC. The glycosylation pattern of each site was determined by MALDI-TOF MS. Our results may provide an insight into the microheterogeneity of glycoproteins. In order to identify the putative flavin-binding site, purified BG60 was limiting digested with trypsin and separated by SDS-PAGE, only the protein band which showed yellow-green fluorescence under UV illumination was subjected to in-gel tryptic digestion. The fluorescent peptide was purified using reverse-phase HPLC by monitoring of excitation at 450 nm and emission at 520 nm; which is the characterization of flavin. N-terminal protein sequence analysis of the fluorescent peptide indicated that the fluorescent compound is linked to His113. However, it was not proved by the result of MALDI-TOF MS and MS/MS analysis that the fluorescent compound purified from BG60 is a flavin. It was suggested that an unknown compound covalently linked to BG60 such as flavin derivatives that might result in similar fluorescent properties as flavin. By combination of enzymatic digestion, HPLC separation and mass spectrometric analysis, we have identified four N-linked glycosylation sites and an unknown fluorescent compound binding site. This strategy may provide an efficient and useful tool for the analysis of post-translational modification sites of protein.