Top-down study of β2-microglobulin deamidation.

Although differentiation of the isomeric Asn deamidation products (Asp and isoAsp) at the peptide level by electron capture dissociation (ECD) has been well-established, isoAsp identification at the intact protein level remains a challenging task. Here, a comprehensive top-down deamidation study is presented using the protein beta2-microglobulin (β(2)M) as the model system. Of the three deamidation sites identified in the aged β(2)M, isoAsp formation was detected at only one site by the top-down ECD analysis. The absence of diagnostic ions likely resulted from an increased number of competing fragmentation channels and a decreased likelihood of product ion separation in ECD of proteins. To overcome this difficulty, an MS(3) approach was applied where a protein ion was first fragmented by collisionally activated dissociation (CAD) and the resulting product ion was isolated and further analyzed by ECD. IsoAsp formation at all three deamidation sites was successfully identified by this CAD-ECD approach. Furthermore, the abundance of the isoAsp diagnostic ion was found to increase linearly with the extent of deamidation. These results demonstrated the potential of ECD in the detection and quantitative analysis of isoAsp formation using the top-down approach.