Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification

Mol Cell Proteomics. 2019 Jan;18(1):16-27. doi: 10.1074/mcp.RA118.000967. Epub 2018 Sep 20.


Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe3+-IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases.

Keywords: Cellular Organelles; Glycoprotein Pathways; Glycoproteins; Glycoproteomics; Lysosomal Disorders; Lysosome; Mannose-6-phosphate; Mannose-6-phosphate Receptors; Phosphorylation; Targeted Degradation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Phosphatase / genetics*
  • Animals
  • Binding Sites
  • CHO Cells
  • Chromatography, Affinity
  • Cricetulus
  • Gene Knockout Techniques
  • Genetic Engineering
  • Glycopeptides / analysis
  • Glycopeptides / chemistry*
  • HeLa Cells
  • Humans
  • Iron / chemistry
  • Lysosomes / metabolism*
  • Mannosephosphates / metabolism*
  • Protein Processing, Post-Translational
  • Proteomics / methods*
  • Tartrate-Resistant Acid Phosphatase / genetics*


  • Glycopeptides
  • Mannosephosphates
  • mannose-6-phosphate
  • Iron
  • ACP2 protein, human
  • ACP5 protein, human
  • Acid Phosphatase
  • Tartrate-Resistant Acid Phosphatase