Title : Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry
Abstract :
- The study of protein glycosylation has lagged far behind the progress of current proteomics because of the enormous complexity, wide dynamic range distribution and low stoichiometric modification of glycoprotein
- Solid phase extraction of tryptic N-glycopeptides by hydrazide chemistry is becoming a popular protocol for the analysis of N-glycoproteome
- However, in silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da
- And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides
- Thus many N-glycosites could not be localized if only trypsin was used to digest proteins
- Herein, we describe a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion
- The lysate of human liver tissue was digested with three proteases, that is, trypsin , pepsin and thermolysin, with different specificities, separately
- Use of trypsin alone resulted in identification of 622 N-glycosites , while using pepsin and thermolysin resulted in identification of 317 additional N-glycosites
- Among the 317 additional N-glycosites , 98 (30.9%) could not be identified by trypsin in theory because the corresponding in silico tryptic peptides are either too small or too big to detect in mass spectrometer
- This study clearly demonstrated that the coverage of N-glycosites could be significantly increased due to the adoption of multiple enzyme digestion
- A total number of 939 N-glycosites were identified confidently, covering 523 noredundant glycoproteins from human liver tissue, which leads to the establishment of the largest data set of glycoproteome from human liver up to now