μLC coupled to ICP–SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification
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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.
KeywordsμLC ICP–MS Isotope dilution Protein quantification CRM 393 CRM 486
The authors are grateful for support from Thermo Fisher Scientific, Bremen. Furthermore, financial support by the DFG within the graduate program GRK 826 “Analysis of element species: development of methods and applications” is gratefully acknowledged. J. Bettmer acknowledges the Spanish ministry for education and science (MEC) for the contract within the Ramón y Cajal program.
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