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Abstract
The amyloid β peptide, as one of the main components in senile plaque, represents a defining pathological feature for Alzheimer’s disease, and is therefore commonly used as a biomarker for this disease in clinical analysis. However, the selection of suitable standards is limited here, since only a few are commercially available, and these suffer from varying purity. Hence, the accurate characterization of these standards is of great importance. In this study, we developed a method for the traceable quantification of the peptide content using species-specific isotope dilution and ICP-MS/MS detection. It is based on the separation of the sulfur-containing amino acids methionine and cysteine after oxidation and hydrolysis of the peptide. Using a strong anion exchange column, both amino acids could be separated from each other, as well as from their oxidized forms and sulfate. The sulfur content was determined via ICP-MS/MS using oxygen as reaction gas. Species-specific isotope dilution was enabled by using a 34S-labeled yeast hydrolysate, containing methionine sulfone and cysteic acid with different isotopic composition. The peptide contents of Aβ standards (Aβ40,42), as well as myoglobin and lysozyme with different degrees of purity, were determined. For validation purposes, the standard reference material NIST 2389a, which contains the amino acids in a similar concentration, was subjected to the developed sample preparation and analysis method. In addition to accounting for errors during sample preparation, high levels of accuracy and precision could be obtained using this method, making it fit-for-purpose for the characterization of peptide standards.Graphical abstract
Highlights
Due to the steady increase in global life expectancy [1], agerelated diseases such as Alzheimer’s disease (AD), the most common form of neurodegenerative disorder [2], are becoming a growing problem
We developed a traceable method for the characterization of protein and peptide standards using speciesspecific isotope dilution by means of HPLC-inductively coupled plasmamass spectrometry (ICP-MS)/MS
A gradient was developed to ensure that the substances elute within the isocratic steps, as the sulfur background increases with higher salt concentration and this could have an impact on the isotope dilution (ID) measurements
Summary
Due to the steady increase in global life expectancy [1], agerelated diseases such as Alzheimer’s disease (AD), the most common form of neurodegenerative disorder [2], are becoming a growing problem. The method employs either isotope-labeled (13C or 15N) amino acids, peptides, or proteins as internal standard, mainly to account for variations in the yield of the analytes from biological samples, where complex matrices can interfere with the measurement. We developed a traceable method for the characterization of protein and peptide standards using speciesspecific isotope dilution by means of HPLC-ICP-MS/MS.
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