title: A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS).
abstract: Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with (34)S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and (34)S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (m(sp)/m(sam)) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5-3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.
authors.affiliation: Division of Metrology in Chemistry, National Institute of Metrology, Beijing, China. Electronic address: fenglx@nim.ac.cn.
authors.forename: Liuxing
authors.initials: L
authors.lastname: Feng
authors.affiliation: Division of Metrology in Chemistry, National Institute of Metrology, Beijing, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Division of Metrology in Chemistry, National Institute of Metrology, Beijing, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Division of Metrology in Chemistry, National Institute of Metrology, Beijing, China.
authors.forename: Dairui
authors.initials: D
authors.lastname: Shen
authors.affiliation: Division of Metrology in Chemistry, National Institute of Metrology, Beijing, China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Li
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007201:Indicator Dilution Techniques; D053685:Laser Therapy; D013058:Mass Spectrometry / Q000379:methods*; D011865:Radioisotope Dilution Technique; D012709:Serum Albumin / Q000032:analysis*; D014168:Transferrin / Q000032:analysis*
chemical_list: D012709:Serum Albumin; D014168:Transferrin
keywords: Albumin; Gel electrophoresis; Inductively coupled plasma mass spectrometry; Laser ablation; Species-unspecific isotope dilution; Transferrin