Characterization of Gel-separated Glycoproteins Using Two-step Proteolytic Digestion Combined with Sequential Microcolumns and Mass Spectrometry

Martin R Larsen,Peter Højrup,Peter Roepstorff

doi:10.1074/mcp.m400068-mcp200

Martin R Larsen, Peter Højrup + Show 1 more

Open Access

https://doi.org/10.1074/mcp.m400068-mcp200

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Abstract

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.

Highlights

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein
By passing the digest through a GELoader tip microcolumn packed with Poros R2 material the unwanted peptides are removed, but the glycopeptides pass through and are trapped on a second GELoader tip microcolumn packed with graphite powder
In the study of ovalbumin with as little as 8 pmol of ovalbumin applied on a gel we identified all of the 13 glycan structures previously observed in HPLC-purified ovalbumin as well as three additional glycan structures

Summary

Introduction

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. This results in small glycopeptides that will have molecular masses typically Ͼ1200 Da. For the phase of the method the remaining non-glycosylated peptides (peptides derived by autoproteolysis of the proteinase K or those resistant to proteinase K) are removed by sequential use of Poros R2 and graphite microcolumns.

Results

Conclusion