Quantification of Structural Alterations of L‐Asp and L‐Asn Residues in Peptides Related to Neuronal Diseases by Reversed‐Phase High‐Performance Liquid Chromatography

Abstract

A method for analyzing the structural alterations in Asn or Asp residues was developed by using the peptides related to neuronal conformational diseases, i.e., the prion protein (PrP)(106-126) and the Alzheimer's amyloid beta (A beta) protein(6-28). The alterations were analyzed by reversed-phase (RP) HPLC, because the peptides containing the structurally altered residues were diastereoisomers of each other, and they were separated with the mobile phase containing an MeCN/sodium phosphate solution and NaCl. The amount of L-Asp, L-isoAsp, D-Asp, or D-isoAsp residues in each PrP peptide isomer was simultaneously quantified by carrying out single HPLC analysis; these residues were generated by the deamidation of the Asn residue. Only 0.3% of the newly generated peptide containing the D-Asp residue was detected. Furthermore, the investigation of the partial fragment of the A beta protein revealed that the present method possessed the ability of simultaneous analysis of the isomerizations of two Asp residues. These results implied that the present method was highly sensitive and reduced the time required for the analysis. This method may accelerate the elucidation of the PrP and A beta protein functions, because the structural alterations of Asn and Asp have been reported to influence these functions.

Determination of rate constants for β-linkage isomerization of three specific aspartyl residues in recombinant human αA-crystallin protein by reversed-phase HPLC

“…The key to our method is the determination of the RP-HPLC separation conditions for the four diastereomers. We established the HPLC conditions for analyzing structurally altered amino acid residues in the peptide fragments of human ␣A-crystallin: 55 TVLDSGISEVR 65 , 79 HFSPEDLTVK 88 , 146 IQTGLDATHAER 157 , and the peptide fragment of the Prion protein, 106 KTNMKHMAGAAAAGAVVGGLG 126 [20,21,26]. The structural alteration of the underlined Asp and Asn residues in these peptides can be quantified by our simple method using RP-HPLC.…”

“…Our present method was based on the separation of peptide diastereomers containing ␣l-, ␣d-, ␤l-, and ␤d-amino acids at specific residues, and succeeded in quantifying the ␤-linkage isomerization and stereoinversion of Asp residues on the peptides related to neuronal diseases such as Alzheimer's disease and Prion disease [26], as well as on the tryptic-digested peptide fragments of ␣A-crystallin protein. Because the spontaneous structural alterations of Asp and Asn residues cause biologically important changes in peptide and protein structures, and function as molecular clocks measuring the time of biological processes such as protein degradation, a simple quantification method using RP-HPLC should greatly facilitate the evaluation of the post-translational modification of a protein and reveal new biological roles for structural alterations at specific amino acids of the protein.…”

Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126

“…69 After peptide synthesis, peptides were purified by RP-HPLC under the isocratic condition (22% acetonitrile containing 0.05% trifluoroacetic acid) and confirmed by the analyses of their amino acid sequences and by mass spectrometry. The peptide solutions were lyophilized and stored at À30 1C until use.…”

Implications of Metal Binding and Asparagine Deamidation for Amyloid Formation

“…Interestingly, the aged PrP, in which Asn 107 is deamidated, forms aggregates and gains proteinase-K resistance in the presence of Cu 2+ . To quantify the structural alteration of Asp residues, we have established a simple method using reverse-phase HPLC, with a standard octadecylsilane column, and applied it to analyze the deamidation of Asn 108 in human PrP 106–126 peptide (or Asn 107 in murine prion) [14]. Under physiological conditions, the Asn 108 is deamidated with a half-life of 10 days (Figure 5A).…”

“…These alterations result in a change in local charge as well as the addition of extra carbon atoms to the polypeptide backbone, which is implicated in various biological phenomena including amyloid fibril formation (Figure 1B). For determining such structural alterations, we have developed a simple method using high-performance liquid chromatography (HPLC), which allowed us to analyze the amount of structurally-altered Asp residues in various proteins, including amyloidogenic peptide fragments [13,14]. …”