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Glycoproteomics: One shot, one pot

Functional Glycomics (08 February 2007) | doi:10.1038/fg.2007.7

A novel approach to glycoproteomics uses a one-pot shotgun strategy to capture and analyze glycoproteins from complex samples.

Profiling of glycoproteins and mapping their glycosylation sites is a difficult task. For a start, the enrichment of glycoproteins from biological samples is complicated by the low abundance of many proteins and the insolubility of native membrane glycoproteins. Tryptic digestion of the proteins, on the other hand, yields a vastly complex mixture of peptides rendering the identification of glycoproteins in low abundance difficult. Sun et al. now report in Molecular and Cellular Proteomics a more sensitive and straightforward approach for comprehensive glycoproteomics than described previously.

The authors first performed a tryptic digestion to solubilize protein samples. Then, as previously proposed, glycosylated peptides were oxidized and captured by immobilized hydrazide resins that exclude non-glycosylated proteins. This lead to a considerable reduction of the complexity of the sample and aided the subsequent mass spectrometric analysis. During the complete procedure, Sun et al. used a one-pot strategy to avoid sample loss. In particular, the use of sodium sulphite as an oxidation quencher replaced the need for a solid-phase capture of oxidized proteins with concomitant sample loss.

After testing the method with simple protein mixtures, the authors applied it to the microsomal fraction of ovarian cancer cells. They identified 302 unique proteins, of which 91% were N-glycosylated. Compared to a general proteomics approach recently applied to the same cancer cell line, the authors found that only 46 proteins overlapped in the two data sets, indicating that both methods are complementary to each other.

The authors also found a higher number of glycosylation sites on identified proteins than the previously reported method. They explain that this is a benefit of tryptic digestion, which by fragmenting proteins improves the solubility of membrane proteins and removes steric hindrances that prevent the capture of inaccessible carbohydrates. The validity of this method is underscored by the identification of many glycoproteins that are already known to be tumor-related proteins of the ovarian cancer cell line.

The authors concentrated on the identification of N-glycosylated proteins but believe that their method can equally be applied to the analysis of O-glycosylations. As opposed to N-glycosylated proteins, they do not possess a consensus sequence for glycosylation, which requires a more elaborate analysis approach. Although the authors say that other methods than mass spectrometry are needed for complete analysis of glycosylation, this method offers a new approach for the glycoproteomic analysis of complex biological samples.

Mirko von Elstermann

Original paper:

  1. Sun B et al. Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics. Mol Cell Proteomics 6, 141–149 (2007). http://www.mcponline.org/cgi/content/abstract/6/1/141 doi: 10.1074/mcp.T600046-MCP200