29 research outputs found
Identification of á´…-amino acid-containing peptides in human serum
<div><p>Biologically uncommon d-aspartate (d-Asp) residues have been shown to accumulate in proteins associated with age-related human disorders, such as cataract and Alzheimer disease. Such d-Asp-containing proteins are unlikely to be broken down completely because metabolic enzymes recognize only proteins or peptides composed exclusively of l-amino acids. Therefore, undigested d-Asp-containing peptides may exist in blood and, if detectable, may be a useful biomarker for associated diseases. In this study, we investigated d-amino acid-containing peptides in adult human serum by a qualitative d-amino acid analysis based on a diastereomer method and LC-MS/MS method. As a result, two d-Asp-containing peptides were detected in serum, both derived from the fibrinogen β-chain, a glycoprotein that helps in the formation of blood clots. One of the peptides was fibrinopeptide B, which prevents fibrinogen from forming polymers of fibrin, and the other was same peptide with C-terminal Arginine missing. To our knowledge, this is the first report of the presence of d-amino acid-containing peptides in serum and the approach described will provide a new direction on the serum proteome and fragmentome.</p></div
Isolation of peptides from human serum.
<p>RP-HPLC chromatogram of the serum peptides from the donor aged ~60 years. The detailed RP-HPLC conditions are described in Materials and Methods. Arrows indicate d-Asp-containing peptides.</p
Identification of d-Asp-containing peptides by LC-MS/MS analysis.
<p>(<b>a</b>) LC-MS chromatogram of peak 16 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189972#pone.0189972.g001" target="_blank">Fig 1</a>. This peptide was separated into two peaks with the same mass ([M+2H]<sup>2+</sup> = 777.5), indicating two isomers of the same peptide. (<b>b-1</b>) Tandem mass spectrum of the large peak in (<b>a</b>). (<b>b-2</b>) Tandem mass spectrum of the small peak in (<b>a</b>). (<b>c</b>) LC-MS chromatogram of peak 18 ([M+2H]<sup>2+</sup> = 698.9) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189972#pone.0189972.g001" target="_blank">Fig 1</a>. (<b>d</b>) Tandem mass spectrum of the peak in (<b>c</b>). Both peptides were identified as fibrinogen β-chain-specific peptides (peak 16, <sup>1</sup>QGVNDNEEGFFSAR<sup>14</sup>; and peak 18, <sup>1</sup>QGVNDNEEGFFSA<sup>13</sup>). The N-terminal Gln residue was converted to pyro-Glu in both peptides.</p
Potential mechanism of isomerization of Asp/Asn in proteins and peptides.
<p>Potential mechanism of isomerization of Asp/Asn in proteins and peptides.</p
Determination of the d/l ratio of Asp in human serum-derived peptides.
<p>Elution profile of the enantiomeric separation of Asp derivatives using Boc-l-Cys-OPA. Aspartate residues from the hydrolysates of peptide peaks 16 (<b>a</b>) and 18 (<b>b</b>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189972#pone.0189972.g001" target="_blank">Fig 1</a> (serum sample from the donor aged ~60 years) are shown. The d/l ratio of Asp was estimated as 0.08 in both peaks by calculating the peak areas of the chromatograms.</p
GM2 levels in the brains of mice.
<p>GM2 levels in brain tissues of wild-type mice (W,♦), untreated Sandhoff mice (S,▪), and Sandhoff mice treated with the modified Hex B (S+E,▴). Each line indicates the mean±SD. *P<0.05 (<i>t</i>-test).</p
HPLC-chromatograms of lyso-GM2 in the brain.
<p>A wild-type mouse (W), an untreated Sandhoff mouse (S), and a Sandhoff mouse treated with the modified Hex B (S+E). Arrows indicate the peak of lyso-GM2.</p
MALDI-TOF-MS spectra of lyso-GM2.
<p>Structure of lyso-GM2 (top) and its spectra obtained for the brain of a Sandhoff mouse (bottom).</p
Plasma lyso-GM2 levels and hexosaminidase activities.
<p>Patient 1, and parents 1-1 and 1-2 are from the same family; patient 3, and parents 3-1 and 3-2 are from the same family; patient 4, and parents 4-1 and 4-2 are from the same family; and patient 5, and parent 5-1 are from the same family.</p
Lyso-GM2 levels in the brains and plasma of mice.
<p>Lyso-GM2 levels in brain tissues (<b>A</b>) and plasma (B). Wild-type mice (W,♦), untreated Sandhoff mice (S,▪), and Sandhoff mice treated with the modified Hex B (S+E,▴). Each line indicates the mean±SD. *P<0.05 (<i>t</i>-test).</p