Ma Jun, Wang Dinghe, She Jessica, Li Jianming, Zhu Jian-Kang, She Yi-Min
Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 201602, China.
Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada.
New Phytol. 2016 Oct;212(1):282-96. doi: 10.1111/nph.14014. Epub 2016 May 12.
N-glycosylation has a great impact on glycoprotein structure, conformation, stability, solubility, immunogenicity and enzyme activity. Structural characterization of N-glycoproteome has been challenging but can provide insights into the extent of protein folding and surface topology. We describe a highly sensitive proteomics method for large-scale identification and quantification of glycoproteins in Arabidopsis through (15) N-metabolic labeling, selective enrichment of glycopeptides, data-dependent MS/MS analysis and automated database searching. In-house databases of Arabidopsis glycoproteins and glycopeptides containing Asn-X-Ser/Thr/Cys motifs were constructed by reducing 20% and 90% of the public database size, respectively, to enable a rapid analysis of large datasets for comprehensive identification and quantification of glycoproteins and heterogeneous N-glycans in a complex mixture. Proteome-wide analysis identified c. 100 stress-related N-glycoproteins, of which the endoplasmic reticulum (ER) resident proteins were examined to be up-regulated. Quantitative measurements provided a molecular signature specific to glycoproteins for determining the degree of plant stress at low temperature. Structural N-glycoproteomics following time-course cold treatments revealed the stress-responsive degradation of high-mannose type N-glycans in ER in response to chilling stress, which may aid in elucidating the cellular mechanisms of protein relocation, transport, trafficking, misfolding and degradation under stress conditions.
N-糖基化对糖蛋白的结构、构象、稳定性、溶解性、免疫原性和酶活性有很大影响。N-糖蛋白质组的结构表征具有挑战性，但能深入了解蛋白质折叠程度和表面拓扑结构。我们描述了一种高灵敏度的蛋白质组学方法，用于通过（¹⁵）N-代谢标记、糖肽的选择性富集、数据依赖型MS/MS分析和自动数据库搜索，对拟南芥中的糖蛋白进行大规模鉴定和定量。通过分别将公共数据库大小减少20%和90%，构建了含有天冬酰胺- X -丝氨酸/苏氨酸/半胱氨酸基序的拟南芥糖蛋白和糖肽内部数据库，以便快速分析大型数据集，全面鉴定和定量复杂混合物中的糖蛋白和异质N-聚糖。全蛋白质组分析鉴定出约100种与胁迫相关的N-糖蛋白，其中内质网（ER）驻留蛋白被检测到上调。定量测量提供了糖蛋白特有的分子特征，用于确定低温下植物胁迫的程度。随时间进程进行冷处理后的结构N-糖蛋白质组学揭示了内质网中高甘露糖型N-聚糖在冷胁迫下的应激反应性降解，这可能有助于阐明胁迫条件下蛋白质重新定位、运输、 trafficking、错误折叠和降解的细胞机制。
