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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Boronate-Based Compounds

Boronic acids bind to cis-diol groups and, thus, are appropriate for extracting a wide range of glycans and glycopeptides. The cyclic compounds that are formed are not particularly stable (binding constants in the region of 103 M), and the bound glycans can easily be released by treatment with mild acids. Like lectins, these boronic acids can be bound to magnetic nanoparticles [41-43] or used to construct affinity columns [44, 45]. In order to increase the binding ability of lectins, Lu et al. [46] have produced boronate-decorated lectins, termed BAD-lectins (ConA, SNA, and Aleuria aurantia lectin (AAL) were used) and conjugated them to magnetic nanoparticles. A 2- to 60-fold increase

in detection sensitivity and enrichment efficiency was observed. 296 N-linked glycopeptides were found in whole-cell lysates with only a 6% overlap between the three lectins.

Hydrazide Enrichment

This method is popular for extracting glycoproteins or glycopeptides, but because the glycan is modified, it is not suitable for structural studies on this part of the molecule. The technique involves oxidation of vicinal diols with periodate to give the corresponding dialdehydes, which are then captured on various media carrying hydrazide groups [47-49]. The proteins are then released, usually with the enzyme protein N-glycosidase F (PNGase F) for further analysis. The oxidized glycan remains attached to the support medium.

Titanium Dioxide Enrichment of Sialylated Glycoproteins

Titanium dioxide is frequently used for enrichment of phosphorylated glycoproteins, but if these are dephosphorylated by treatment with alkaline phosphatase, the titanium dioxide binds highly efficiently to any sialic acids [50, 51]. Nonspecific binding of acidic amino acids can be blocked with 5% trifluoro- acetic acid (TFA) and 1 M glycolic acid. The bound glycoproteins can then be released by treatment with aqueous ammonia at pH 11.

 
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