Rapid Survey of Four Asp Isomers in Disease-Related Proteins by LC-MS combined with Commercial Enzymes
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- 作者：Hiroki Maeda ; Takumi Takata ; Norihiko Fujii ; Hiroaki Sakaue ; Satoru Nirasawa ; Saori Takahashi ; Hiroshi Sasaki ; Noriko Fujii
- 刊名：Analytical Chemistry
- 出版年：2015
- 出版时间：January 6, 2015
- 年：2015
- 卷：87
- 期：1
- 页码：561-568
- 全文大小：443K
- ISSN：1520-6882
文摘
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伪, l尾, d尾, and d伪) 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伪-Asp residues, it differentiates peptides containing l伪-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing l尾-Asp residues, and paenidase internally cleaves the C-terminus of d伪-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.