Development of quantitative mass spectrometric immunoassay for serum amyloid A

Abstract

Objective: Proteins can exist as multiple proteoforms in vivo that can have important roles in physiological and pathological states.

Methods: We present the development and characterization of mass spectrometric immunoassay (MSIA) for quantitative determination of serum amyloid A (SAA) proteoforms.

Results: Intra- and inter-day precision revealed CVs <10%. Against existing SAA ELISA, the developed MSIA showed good correlation according to the Altman-Bland plot. Individual concentrations of the SAA proteoforms across a cohort of 170 samples revealed 7 diverse SAA polymorphic types and 12 different proteoforms.

Conclusion: The new SAA MSIA enables parallel analysis of SAA polymorphisms and quantification of all expressed SAA proteoforms, in a high-throughput and time-efficient manner.

Keywords: Baseline concentration; inflammation biomarker; mass spectrometry; polymorphism; posttranslational modifications; proteoforms.

Fig. 1

a) Example SAA standard curve with BL as an IRS; b) Representative mass spectra from generated standard curve, showing peaks for SAA and BL in different SAA concentrations.

Fig. 1

a) Example SAA standard curve with BL as an IRS; b) Representative mass spectra from generated standard curve, showing peaks for SAA and BL in different SAA concentrations.

Fig. 2

SAA method validation MSIA vs ELISA a) scatter plot (dashed line represents the obtained regression; dotted line represents identity (x=y); b) Bland-Altman difference plot (solid line represents the mean of concentration difference, 2 mg/L; dashed lines represent limits of agreement ±1.96 SD).

Fig. 2

SAA method validation MSIA vs ELISA a) scatter plot (dashed line represents the obtained regression; dotted line represents identity (x=y); b) Bland-Altman difference plot (solid line represents the mean of concentration difference, 2 mg/L; dashed lines represent limits of agreement ±1.96 SD).

Fig. 3

Example mass spectra of serum amyloid A and beta lactoglobulin extracted from human plasma samples using MSIA. *The inlets in the mass spectra represent close-ups of three different SAA protein profiles from three human plasma samples. **Signals labeled with an asterisks (*) represent MALDI matrix adducts.

Fig. 4

Total SAA concentration distribution among different SAA polymorphic profiles identified in the sample cohort; * The inlets present the percentage distribution of the SAA polymorphic profiles (left) and the total average SAA concentration in each sample subset (right).

Fig. 5

Concentration distribution of posttranslationally modified SAA proteoforms: a) average total concentrations of SAA 1.1 proteins; b) average total concentrations of SAA 1.3 proteins; c) average total concentrations of SAA 2.1 proteins; d) average total concentrations of SAA 2.2 proteins.