Enrichment Strategies

Enrichment methods to facilitate detection and quantitation of PTM proteins are a common strategy in proteomics. A boronate affinity chromatography method has been used for the fructosamine proteome based on the binding of the cis-1,2-diol structure of fructosamine-modified proteins, with subsequent release from the boronate affinity matrix with weak acid. Although some enzymatically glycosylated proteins contain cis-1,2-diol moieties, steric effects, proximate negatively charged groups, and acetylation limit the retention and interference in this method by glycoproteins [98]. A similar affinity method is used in the routine separation of hemoglobin in clinical chemistry to quantify glycated hemoglobin HbA1c for the assessment of glycemic control in diabetes [99]. Boronate affinity enrichment of protein glycated by glucose was employed in a study of glycated proteins in human plasma and red blood cells, and 7749 unique glycated peptides corresponding to 3742 unique glycated proteins were identified [94].

The dihydroxyimidazolidine residues present in proteins glycated by MG and glyoxal are also a potential interference in boronate affinity chromatography for the enrichment of proteins as they contain a side chain with a cis-1,2-diol moiety [100]. This has been exploited to identify proteins with arginine residues activated for reaction with glyoxal derivatives. Reaction of proteins with butane-2,3-dione formed 4,5-dihydroxy-4,5-dimethylimidazolidine residues of proteins containing activated arginine residues. Proteins with such residues on the surface were retained in boronate affinity chromatography [101, 102].

In principle antibodies to fructosamine and hydroimidazolones may be used for immunoaffinity purification and enrichment of proteins glycated by glucose and MG, respectively. Limited specificity of antifructosamine and anti-AGE antibodies, as indicated by poor performance in immunoassay [2], suggests that this is not currently a reliable enrichment strategy. Where immu- noaffinity enrichment is employed, it is vital to confirm the presence of a gly- cation adduct residue in the retained proteins by mass spectrometric analysis.

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