Integrative Quantitative Proteomics and N-Glycoproteomics Reveal Age-Processing of N-Glycosylation in Caenorhabditis elegans.

N-glycosylation, a highly diverse post-translational modification, critically regulates organismal aging. The Caenorhabditis elegans (C. elegans) model is one of the excellent aging models, which exhibits distinctive N-glycosylation features including highly fucosylated core structures, bisecting β1,4-galactose, extensive phosphocholine, and methyl modifications. However, the site-specific and structure-specific landscape of the intact N-glycoproteome in adult nematodes is still unknown. In this study, we generated the first temporal atlas of the N-glycoproteome across the three stages of young adulthood (day 1), mid-life (day 5), and early senescence (day 10) of C. elegans, which identified 369 distinct glycoforms at 196 N-glycosites on 161 glycoproteins. A mixed sparse Partial Least Squares Discriminant Analysis (sPLS-DA) model, built from dual-omics data, decoded proteins and N-glycoproteins may act synergistically during the adult age progression. Time-series analysis further elucidated four distinct glycoprotein expression patterns and their functional implications. This represents the largest temporal N-glycosylation dataset of C. elegans to our knowledge. A high-throughput identification and quantification pipeline for the intact N-glycopeptides of C. elegans has been established. This study highlights key N-glycoproteins and glycans implicated in the regulation of adult age progression and may contribute to the integrative analysis of N-glycoproteome data with other omics datasets.