Procedure providing SI-traceable results for the calibration of protein standards by sulfur determination and its application on tau

Abstract

Quantitative proteomics is a growing research area and one of the most important tools in the life sciences. Well-characterized and quantified protein standards are needed to achieve accurate and reliable results. However, only a limited number of sufficiently characterized protein standards are currently available. To fill this gap, a method for traceable protein quantification using sulfur isotope dilution inductively coupled plasma mass spectrometry (ICP-MS) was developed in this study. Gel filtration and membrane filtration were tested for the separation of non-protein-bound sulfur in the protein solution. Membrane filtration demonstrated a better performance due to the lower workload and the very low sulfur blanks of 11 ng, making it well suited for high-purity proteins such as NIST SRM 927, a bovine serum albumin (BSA). The method development was accomplished with NIST SRM 927e and a commercial avidin. The quantified mass fraction of NIST SRM 927e agreed very well with the certified value and showed similar uncertainties (3.6%) as established methods while requiring less sample preparation and no species-specific standards. Finally, the developed procedure was applied to the tau protein, which is a biomarker for a group of neurodegenerative diseases denoted "tauopathies" including, e.g., Alzheimer's disease and frontotemporal dementia. For the absolute quantification of tau in the brain of transgenic mice overexpressing human tau, a well-defined calibration standard was needed. Therefore, a pure tau solution was quantified, yielding a protein mass fraction of (0.328 ± 0.036) g/kg, which was confirmed by amino acid analysis.

Keywords: Inductively coupled plasma mass spectrometry; Isotope dilution; Quantitative protein analysis; SI traceability; Sulfur.

Fig. 1

Calculation scheme for the correction of the sulfur mass fraction of a protein sample for non-protein-bound sulfur after separation by membrane filtration

Fig. 2

Contributors to the uncertainty of the avidin mass fraction. R_x(Protein, CIAAW) and x_x,b(Protein, CIAAW) were taken from tabulated data published by CIAAW. The quantities are given as intervals, and uncertainties were determined as rectangular functions of these intervals. The molar mass of avidin M(Avidin, Uniprot) was taken from the Uniprot database, and a high uncertainty of 5% was estimated for this quantity

Fig. 3

Metrological traceability chain and calibration hierarchy according to IUPAC guidelines [46], shown on the example of BSA sample 1–1