Abstract
BackgroundGlycosylation 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.Methods/Principal FindingsA 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 was developed recently and we optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. We 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.Conclusions/SignificanceWe demonstrate, for the first time, heterogeneous O-glycosylation profile of a protein by a whole protein analysis using MALDI-MS. 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.
Highlights
Glycosylation is a common and highly diverse co- and posttranslational protein modification [1]
Peptides of different hydrophilicity can be separated on the liquid matrix 3AQ/CHCA spotted onto a MALDI target plate [19]
An ontarget separation method, making use of the liquid matrix 3AQ/ CHCA for MALDI-MS, was developed recently and applied to analyze N-glycosylation of ribonuclease B
Summary
Glycosylation is a common and highly diverse co- and posttranslational protein modification [1]. Accumulating evidence indicates that addition or modification of one or more sugar moieties can have diverse effects on protein function and such modifications have been implicated in different types of disease [2]. O-glycosylation is initiated by the addition of an N-acetylgalactosamine (GalNAc) to a hydroxyl group on a serine or threonine residue followed by the subsequent addition of galactose or N-acetylglucosamine (GlcNAc), which forms the core structure of the glycan chain [6]. It has been reported that the core structures of O-glycans are involved in the metastatic capacity of cancer cells. Simple and rapid methods to analyze glycans on individual proteins have not been available until recently
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