Dual-Functional Titanium (IV) Immobilized Metal Affinity Chromatography Enables Large-scale Profiling of Protein Mannose-6-Phosphate Glycosylation and Reveals Its Predominant Substrates

• Published in: Analytical chemistry (Washington) Vol. 91; no. 18; pp. 11589 - 11597
• Main Authors: Huang, Junfeng, Dong, Jing, Shi, Xudong, Chen, Zhengwei, Cui, Yusi, Liu, Xiaoyan, Ye, Mingliang, Li, Lingjun
• Format: Journal Article
• Language: English
• Published: 22.08.2019
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.9b01698

Mannose-6-phosphate (M6P) glycosylation is an important post-translational modification (PTM) and plays a crucial role in transferring lysosomal hydrolases to lysosome and is involved in several other biological processes. Aberrant M6P modifications have been implicated in lysosomal storage diseases and numerous other disorders including Alzheimer’s disease and cancer. Research on profiling of intact M6P glycopeptides remains to be challenging due to its extremely low stoichiometry. Here we propose a dual-mode affinity approach to enrich M6P glycopeptides by dual-functional titanium (IV) immobilized metal affinity chromatography (Ti(IV)-IMAC) materials. In combination with state-of-the-art mass spectrometry and database search engine, we profiled 237 intact M6P glycopeptides corresponding to 81 M6P glycoproteins in five types of tissues in mouse, representing the first large-scale profiling of M6P glycosylation in mouse samples. The analysis of M6P glycoforms revealed the predominant glycan substrates of this PTM. Gene ontology analysis showed that overrepresented M6P glycoproteins were lysosomal associated proteins. However, there were still substantial M6P glycoproteins that possessed different subcellular locations and molecular functions. Deep mining of their roles implicated in lysosomal and non-lysosomal function can provide new insights into functional roles of this important yet poorly studied modification.