Rapid Survey of Four Asp Isomers in Disease-Related Proteins by LC-MS combined with Commercial Enzymes

Until relatively recently, it was considered that d-amino acids were excluded from living systems except for the cell wall of microorganisms. However, d-aspartate residues have now been detected in long-lived proteins from various tissues of elderly humans. Formation of d-aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent study used LC-MS to analyze isomers of Asp residues in proteins precisely without complex purification of the proteins. However, to identify the four Asp isomers (L alpha, L beta, D beta, and D alpha) on the chromatogram, it was necessary to synthesize reference peptides containing the four different Asp isomers as standards. Here, we describe a method for rapidly and comprehensively identifying Asp isomers in proteins using a combination of LC-MS and commercial enzymes without synthesizing reference peptides. The protein sample is treated with trypsin, trypsin plus Asp-N, trypsin plus PIMT, trypsin plus paenidase, and the resulting peptides are applied to LC-MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only L alpha-Asp residues, it differentiates peptides containing L alpha-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing L beta-Asp residues, and paenidase internally cleaves the C-terminus of da-Asp residues. This approach was successfully applied to the analysis of all tryptic peptides in aged lens. The comprehensive quantitative data of Asp isomer formation in age-related proteins obtained via this method might be used as biomarkers of age-related disease.