μLC coupled to ICP–SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification
| Autor: | Ralf Krüger, Jörg Bettmer, Nico Zinn, Peter Leonhard |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Radioisotope Dilution Technique
Spectrometry Mass Electrospray Ionization Bioanalysis Chromatography Apolipoprotein A-I Elution Chemistry Analytical chemistry Proteins Isotope dilution Mass spectrometry Biochemistry Analytical Chemistry Certified reference materials Limit of Detection Sulfur Isotopes Animals Humans Amino Acid Sequence alpha-Fetoproteins Inductively coupled plasma Quantitative analysis (chemistry) Inductively coupled plasma mass spectrometry Chromatography High Pressure Liquid |
| Zdroj: | Analytical and Bioanalytical Chemistry. 391:537-543 |
| ISSN: | 1618-2650 1618-2642 |
| DOI: | 10.1007/s00216-008-2025-9 |
| Popis: | Absolute protein quantification has become an important challenge in modern bioanalytical chemistry. Among several approaches based on mass spectrometric techniques, inductively coupled plasma (ICP) as ionisation source provides element-selective and sensitive detection of heteroatoms, and thus, a potentially emerging tool in protein analysis. In this work we applied coupling of capillary liquid chromatography (μLC) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) to the separation and determination of standard proteins. For quantification purposes, post-column isotope dilution of sulfur was applied and optimised for this type of hyphenated technique. Provided that the protein sequence is known (number of sulfur-containing amino acids, i.e. cysteines and methionines) the protein amount can then be directly calculated from the determined sulfur content in a certain protein fraction. In order to prove the reliability of the presented method, two different certified reference materials were analysed: CRM 393 (human apolipoprotein A-I) and CRM 486 (α-fetoprotein). For CRM 393 excellent agreement (37.0 ± 1.4 μmol L(-1)) was obtained with the certificate (37.7 ± 1.8 μmol L(-1)). However, the recovery rate for α-fetoprotein in CRM 486 was found to be about 60% indicating incomplete elution of the protein during the chromatographic separation. |
| Databáze: | OpenAIRE |
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