Detection of the Heterogeneous O-Glycosylation Profile of MT1-MMP Expressed in Cancer Cells by a Simple MALDI-MS Method



Glycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently.

In present study authors developed a new technique to analyze glycopeptides in a highly sensitive manner by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the liquid matrix 3AQ/CHCA and  optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. They used the MALDI-MS method to evaluate glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP). O-glycosylation of MT1-MMP is reported to modulate its protease activity and thereby to affect cancer cell invasion.

MT1-MMP expressed in human fibrosarcoma HT1080 cells was immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest was applied directly to the liquid matrix on a MALDI target plate. Concentration of hydrophilic glycopeptides within the central area occurred due to gradual evaporation of the sample solution, whereas nonglycosylated hydrophobic peptides remained at the periphery. This specific separation and concentration of the glycopeptides enabled comprehensive analysis of the MT1-MMP O-glycosylation.

Since cancer cells are reported to have altered glycosylation of proteins, this easy-to-use method for glycopeptide analysis opens up the possibility to identify specific glycosylation patterns of proteins that can be used as new biomarkers for malignant tumors.




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