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.

The postnatal presence of human chorionic gonadotropin in preterm infants and its potential inverse association with retinopathy of prematurity

“…Considering the miniscule amounts of infant hormone that would be available for analysis and the small sample volume (from 3 mm punches from DBS), we determined that mass spectrometry would not be a viable analytical method for this study. (35,36) Alternatively, we opted to utilize ECL technology on the MSD multi-plex platform since it provides exquisite detection sensitivity with reduced total amount of analyte required; (27,37) moreover, it has been successfully used in prior neonatal DBS analyses. 28 Another limitation of the study was the limited information available for each patient due to use of precollected, de-identified samples.…”

Matrix‐assisted ionization mass spectrometry in targeted protein analysis – An initial evaluation

“…To test our hypothesis, we evaluated whether previously established ESI-SRM-MS-settings 22,31,32 could be directly applied to perform MAI-SRM-MS. Standard MAI conditions 11 were used: a matrix FIGURE 1 Photograph of the commercial Nanosource flex™ ion source: A, unmodified and B, after stripping providing direct access to the mass spectrometer inlet to perform MAI-MS. Briefly, 3-NBN matrix dissolved in acetonitrile and aqueous analyte solution are combined in a 1:3 (v/v) ratio and 1 μL, preferably crystallized outside the syringe tip, is brought into close proximity to the mass spectrometer inlet where the sample is entrapped into the sub-atmospheric pressure where ionization commences 13 [Color figure can be viewed at wileyonlinelibrary.com] consisting of 3-NBN in acetonitrile, and peptides at 5 μM in water (volume ratio 3:1) without optimization using the syringe introduction 16 but without penetrating the tip into the inlet.…”

“…Overview of transitions and collision energies for the tryptic peptides and SIL-peptides used for MAI-SRM-MS in analogy to ESI-SRM-MS22,31,32 …”

Antibody based affinity capture LC-MS/MS in quantitative determination of proteins in biological matrices

“…Since the (reagent) antibody will interact with only a relative small part of a protein, changes in the target protein, which are neither within nor close to the epitope region, will not affect the immune-reactivity of the reagent antibody. This allows capture of target protein isoforms and classes of proteins [29], and up to 8 variants of hCG in human urine and serum [30]. The quantification of 1-84 parathyroid hormone in patient samples at pM levels by immuno-capture LC-MS showed its superiority over conventional immunoassays with respect to the ability to distinguish and determine C-terminal metabolic peptide products [31].…”

“…Although the variations on the primary protein structure level can nicely be detected by determining their proteotypic peptides in the bottom-up approach, it will not be possible to determine if a particular proteotypic peptide originates from the β-subunit in the hetero-dimer or from the β-subunit in the monomer (variations in the quaternary protein structure). Sequential extractions for hCG were developed to enable determination of variations in the primary protein structure and quaternary protein structure in urine [50] and serum samples [30]. In the urine example first the monomers were extracted followed by the intact heterodimer, in the serum example the order of extraction was reversed.…”