ANYL 380 |
| Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. When isoaspartate forms, a methylene group is inserted into the protein back bone. This modification has been shown to drastically, in many cases adversely, alter protein structure and function. For instance, isoaspartate accumulation has been associated with aging and amyloid formation, and isoaspartate formation is a major contributing factor to the heterogeneity observed in protein pharmaceutics. Currently isoaspartyl proteins or peptides are detected by immunological, instrumental and enzymatic methods. Instrumental methods (such as HPLC and IEF) are most commonly used, but cannot directly identify the isoaspartate containing analytes. We report here a chemo-enzymatic detection method for protein isoaspartate. In the initial step, protein isoaspartate methyltransferase (PIMT) selectively converts isoaspartyl residues into the corresponding methyl esters, which are intrinsically labile. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The hydrazide end products are stable and can be analyzed by various proteomic techniques, such as MALDI and ESI mass spectrometry. For example, the precise location of these isoaspartate residues was determined by tandem mass spectroscopic analysis. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as ultraviolet-visible detection through a dansyl-derivative. Alternatively, the hydrazide product can be enriched by affinity chromatography, thus enhancing analyte signal and simplifying product identification. Our chemo-enzymatic method reported here should be amendable for isoaspartate analysis in proteomic research and protein pharmaceutics characterization. |
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Analytical Approaches
9:00 AM-12:00 PM, Wednesday, August 22, 2007 BCEC -- 104B, Oral
Division of Analytical Chemistry |