Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.
Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.
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DOI：
10.1021/pr2001429
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发表时间：
2011-05-06
影响因子：
4.4
通讯作者：
Lebrilla, Carlito B.
中科院分区：
文献类型：
--
作者：
Nwosu, Charles C.;Seipert, Richard R.;Strum, John S.;Hua, Serenus S.;An, Hyun Joo;Zivkovic, Angela M.;German, Bruce J.;Lebrilla, Carlito B.
关键词：
由于蛋白质中糖基化的性质和复杂性，单个糖蛋白的广泛位点特异性糖基化分析是困难的。在蛋白质混合物中，这些分析甚至更加困难。我们提出了一种结合非特异性蛋白酶消化，纳米流液相色谱和串联质谱（MS/MS）的方法，旨在全面的蛋白质混合物中的位点特异性糖基化分析。本文所述的策略涉及使用与四极杆飞行时间质谱联用的纳米流液相色谱法（nano-LC/Q-TOF MS）分析由固定化链霉蛋白酶消化由牛乳铁蛋白、κ酪蛋白和牛胎球蛋白组成的糖蛋白混合物产生的糖肽的复杂混合物。所得糖肽在填充有多孔石墨化碳的微流体芯片上进行色谱分离，并通过MS和MS/MS分析进行分析。总之，在单一混合物中观察并测定了对应于18个糖位点的233种糖肽（根据组成鉴定，包括异构体）。糖肽是N-连接糖肽（含有高甘露糖、复合和杂合聚糖）和O-连接糖肽（主要是唾液酸化）的混合物。由于获得了详细的聚糖微观异质性信息，因此本研究的结果是全面的。这种方法提供了一个平台，同时表征N-和O-糖基在同一混合物中广泛的网站异质性。
Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining non-specific protease digestion, nano-flow liquid chromatography and tandem mass spectrometry (MS/MS) aimed at comprehensive site-specific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein and bovine fetuin using nano-flow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan micro-heterogeneity information was obtained. This approach presents a platform to simultaneously characterize N- and O-glycosites in the same mixture with extensive site heterogeneity.
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影响因子：
3.4
作者：
Holland, JW;Deeth, HC;Alewood, PF
通讯作者：
Alewood, PF
影响因子：
7.4
作者：
Medzihradszky, KF;Besman, MJ;Burlingarne, AL
通讯作者：
Burlingarne, AL
影响因子：
7.4
作者：
An, HJ;Peavy, TR;Lebrilla, CB
通讯作者：
Lebrilla, CB
影响因子：
3.1
作者：
CODDEVILLE, B;STRECKER, G;SPIK, G
通讯作者：
SPIK, G
在微流体芯片和飞行时间质谱上使用高性能液相色谱法的天然N连接聚糖结构的剖面。
影响因子：
3.4
作者：
Chu, Caroline S.;Ninonuevo, Milady R.;Clowers, Brian H.;Perkins, Patrick D.;An, Hyun Joo;Yin, Hongfeng;Killeen, Kevin;Miyamoto, Suzanne;Grimm, Rudolf;Lebrilla, Carlito B.
通讯作者：
Lebrilla, Carlito B.