A New Strategy for High-Efficient Tandem Enrichment and Simultaneous Profiling of N-Glycopeptides and Phosphopeptides...

N-glycosylation and phosphorylation, two common posttranslational modifications, play important roles in various biological processes and are extensively studied for biomarker and drug target screening. Because of their low abundance, enrichment of N-glycopeptides and phosphopeptides prior to LC-MS/MS analysis is essential. However, simultaneous characterization of these two types of posttranslational modifications in complex biological samples is still challenging, especially for tiny amount of samples obtained in tissue biopsy. Here, we introduced a new strategy for the highly efficient tandem enrichment of N-glycopeptides and phosphopeptides using HILIC and TiO₂ microparticles. The N-glycopeptides and phosphosites obtained by tandem enrichment were 21%-377% and 22%-263% higher than those obtained by enriching the two PTM peptides separately, respectively, using 160-20 μg tryptic digested peptides as the starting material. Under the optimized conditions, 2798 N-glycopeptides from 434 N-glycoproteins and 5130 phosphosites from 1986 phosphoproteins were confidently identified from three technical replicates of HeLa cells by mass spectrometry analysis. Application of this tandem enrichment strategy in a lung cancer study led to simultaneous characterization of the two PTM peptides and discovery of hundreds of differentially expressed N-glycosylated and phosphorylated proteins between cancer and normal tissues, demonstrating the high sensitivity of this strategy for investigation of dysregulated PTMs using very limited clinical samples.