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

Abstract

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 (Lα, Lβ, Dβ, and Dα) 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 Lα-Asp residues, it differentiates peptides containing Lα-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing Lβ-Asp residues, and paenidase internally cleaves the C-terminus of Dα-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.

Establishment of a Two-Dimensional HPLC-MS/MS Method Combined with DCl/D₂O Hydrolysis for the Determination of Trace Amounts of D-Amino Acid Residues in Proteins

“…An enantioselective sequencing method combining Edman degradation and enantioselective analysis of the produced PTH amino acids has also been reported [18]. For the screening of diastereomeric peptides containing D-amino acids, the use of an LC-MS/MS method is also effective [17]. However, these analytical methods are complicated and only applicable to selected sequences of peptides mainly containing Asp residues.…”

“…Normally, the proteins were enzymatically digested, and the peptides containing the target D-amino acid residues are determined as diastereomers [16][17]. Edman degradation combined with enantioselective analysis of the produced PTH amino acids has also been reported [18].…”

Isomerization of aspartyl residues in crystallins and its influence upon cataract

“…To eliminate the synthesis of reference peptides and reduce the analytical time, we therefore developed an advanced method for rapidly identifying Asp isomers in proteins based on a combination of LC-MS/ MS and commercial enzymes [34]. In this approach, the protein sample is treated with trypsin, trypsin plus endoprotease Asp-N (Asp-N), trypsin plus L-isoaspartyl methyltransferase (PIMT) or trypsin plus paenidase, and the resulting peptides are applied to LC-MS/MS.…”

Enantiomeric Ratio of Amino Acids as a Tool for Determination of Aging and Disease Diagnostics by Chromatographic Measurement

“…The distribution and physiological role of D-AAs during normal aging, occurrence of D-AAs in pathology, relation between D-AAs and conformational diseases of brain, and some methods for the determination of D-AAs and their metabolites are also included. D-aspartyl residues were detected in various proteins from diverse tissues of elderly individuals [38,39]. Moreover, detection of D-β-Asp is associated with some age-related diseases.…”

“…The comprehensive and quantitative data of Asp isomer formation (Lα, Lβ, Dβ, Dα) in age-related proteins might be used as a reliable biomarker for age-related diseases such as Alzheimer's disease (AD), cataracts, etc. [39,40]. Ultra-high performance liquid chromatography methods for the determination and analysis of disease biomarkers including some chiral methods for AAs were clearly reviewed [41].…”