title: μLC coupled to ICP-SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification.
abstract: 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.
authors: Molecular Structure Analysis, German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors: Nico
authors: N
authors: Zinn
authors: Ralf
authors: R
authors: Krüger
authors: Peter
authors: P
authors: Leonhard
authors: Jörg
authors: J
authors: Bettmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D011506:Proteins / Q000032:analysis*; D011865:Radioisotope Dilution Technique; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods; D013460:Sulfur Isotopes / Q000032:analysis*; D000509:alpha-Fetoproteins / Q000032:analysis
chemical_list: C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D011506:Proteins; D013460:Sulfur Isotopes; D000509:alpha-Fetoproteins