Integration of high-pH fractionation and cotton-HILIC enrichment approaches for analyzing N-glycopeptides

Tahhan N., Aksoy S., Kayili H. M., SALİH B.

Advances in Sample Preparation, vol.8, 2023 (Scopus) identifier

  • Publication Type: Article / Article
  • Volume: 8
  • Publication Date: 2023
  • Doi Number: 10.1016/j.sampre.2023.100098
  • Journal Name: Advances in Sample Preparation
  • Journal Indexes: Scopus
  • Keywords: Cotton-HILIC, Enrichment strategies, Fractionation, Glycoproteomics, Mass spectrometry, N-glycosylation
  • Hacettepe University Affiliated: Yes


Glycosylation, a prevalent post-translational modification, plays a significant role in diverse biological processes and exhibits associations with various diseases, including cancer. Among the different types of glycosylation, N-glycosylation is particularly prominent. Glycosylation analysis also plays a crucial role in understanding the efficacy of protein biopharmaceuticals, as the majority of these drugs are glycoproteins. The precise detection and characterization of N-glycosylation require a series of enrichment steps. On the other hand, peptide fragmentation methods constitute a crucial step in reducing sample complexity. Moreover, enrichment strategies coupled with mass spectrometry are essential for the analysis and identification of N-glycopeptides. This research aims to enhance the detection methods for N-glycopeptides and N-glycoproteins by employing strategies that involve peptide fractionation and glycopeptide enrichment. This study targeted to assess and compare the efficacy of an integration method and a direct enrichment approach in terms of identifying glycopeptides from the glycoproteome of human plasma. The results demonstrate that the integration method detects 212 N-glycopeptides and 88 N-glycoproteins, while Cotton-HILIC direct enrichment detects 88 N-glycopeptides and 41 N-glycoproteins. In conclusion, the employment of Cotton-HILIC enrichment with high pH fractionation exhibits greater qualitative abilities in characterizing the structures and functions of glycopeptides.