title: Dual-immuno-MS technique for improved differentiation power in heterodimeric protein biomarker analysis: determination and differentiation of human chorionic gonadotropin variants in serum.
abstract: If the biomarker potential of intact heteromers and their free subunits is different, differentiation between these forms may reveal important clinical information. Such differentiation may however be analytically challenging. One possible way of circumventing this challenge is by performing a dual-immuno-MS approach. In the present paper, a two-step immunoaffinity sample preparation step is succeeded by digestion and subsequent LC-MS analysis to provide high-sensitivity quantification and differentiation between the heterodimer human chorionic gonadotropin (hCG) and its free β-subunit in serum. Intact and free variants are captured in two separate immunoextraction steps in order to increase the differentiation power of the method. Intact heterodimer variants were depleted prior to free subunit variants in order to incorporate a method quality control. The method was optimized for serum samples. A fully validated immuno-MS method was used as foundation, and partial validation according to the European Medicines Agency's (EMA) guidelines on validation of bioanalytical methods was performed for the dual approach. An accelerated digestion step was incorporated making batch processing of samples within 1 day possible (approx. 3.5 h of sample preparation including digestion). Acceptable linearity (R (2) ≥ 0.990 for four variants and R (2) of 0.920 and 0.966 for the remaining two) and specificity were demonstrated, and the method was robust toward varying levels of intact heterodimer versus free subunit. The method was also successfully tested on realistic samples, demonstrating both the differences in total hCG and the distribution between intact hCG and its free β-subunit in real samples. Graphical abstract Schematic overview of the dual immuno-MS process.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Siri Valen
authors.initials: SV
authors.lastname: Egeland
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Léon
authors.initials: L
authors.lastname: Reubsaet
authors.affiliation: Department of Medical Biochemistry, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Paus
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway. trinegha@farmasi.uio.no.
authors.forename: Trine Grønhaug
authors.initials: TG
authors.lastname: Halvorsen
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D006063:Chorionic Gonadotropin / Q000032:analysis / Q000097:blood*; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000032:analysis / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007163:Immunosorbent Techniques; D057230:Limit of Detection; D055503:Protein Multimerization; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human
keywords: Biomarkers; Differentiation power; Heterodimers; Immunocapture; LC-MS/MS; Proteins