title: Endoplasmic reticulum-associated N-glycan degradation of cold-upregulated glycoproteins in response to chilling stress in Arabidopsis.
abstract: 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.
authors: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 201602, China.
authors: Jun
authors: J
authors: Ma
authors: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 201602, China.
authors: Dinghe
authors: D
authors: Wang
authors: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 201602, China.|Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada.
authors: Jessica
authors: J
authors: She
authors: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 201602, China.
authors: Jianming
authors: J
authors: Li
authors: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 201602, China.
authors: Jian-Kang
authors: JK
authors: Zhu
authors: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 201602, China.
authors: Yi-Min
authors: YM
authors: She
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D003080:Cold Temperature; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005615:Freezing; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D015203:Reproducibility of Results; D013312:Stress, Physiological; D015854:Up-Regulation
chemical_list: D029681:Arabidopsis Proteins; D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides
keywords: Arabidopsis; chilling stress; endoplasmic reticulum (ER); glycoproteomics; liquid chromatography-tandem mass spectrometry