Identification of Isomeric Aspartate residues in βB2-crystallin from Aged Human Lens
| Autor: | Atsuhiko Toyama, Takumi Takata, Kento Murakami, Noriko Fujii |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Aging Stereochemistry Biophysics Peptide Stereoisomerism Biochemistry Analytical Chemistry Lens protein 03 medical and health sciences Crystallin Tandem Mass Spectrometry Aspartic acid Lens Crystalline beta-Crystallin B Chain Humans Deamidation Molecular Biology Chromatography High Pressure Liquid Aged chemistry.chemical_classification Aspartic Acid Chromatography Reverse-Phase 030102 biochemistry & molecular biology Amino acid 030104 developmental biology chemistry sense organs Isomerization |
| Zdroj: | Biochimica et biophysica acta. Proteins and proteomics. 1866(7) |
| ISSN: | 1570-9639 |
| Popis: | Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as β- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in β- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens βB2-crystallin, the main component of the β-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in βB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of βB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of βB2-crystallin and thus likely to have an impact on crystallin subunit-subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens. |
| Databáze: | OpenAIRE |
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