Endoplasmic reticulum‐associated N‐glycan degradation of cold‐upregulated glycoproteins in response to chilling stress in <i>Arabidopsis</i>

Summary 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.