Since 2015, Quality Assistance proposes to its partners a method based on Sulphur specific ICP-MS/MS detection combined with an 34S/32S isotope dilution quantification. Over the years, high precision and accuracy of this methodology gained recognition; however, the sample quantity required by the method was limiting its applicability, especially for early-stage projects. Thanks to the expertise gained over the application of the method, it was redesigned to decrease by ten-fold the sample uptake. This application note presents an overview of this optimised method together with a demonstration of its performance through a partial validation study. This may be considered as a state-of-the-art approach for absolute quantification of proteins, to be used for both biological reference standard qualification and for protein molecular extinction coefficient determination.
Excellent accuracy and precision of the method were demonstrated for most of the tested conditions, meeting mean %Recoveries between 100-102%, except for matrix spiked with higher levels of methionine for which obtained mean %Recoveries were between 102 – 103%. All % RSDs measured were below 1.0%. Moreover, sample uptake for this optimised method is as low as 4 mg of protein (total quantity required for the triplicate preparation).
The partial validation of the method was carried out in accordance with ICH recommendations. The method shows excellent capabilities for accurate protein determination in the presence of challenging matrices. Therefore, it can be considered as a generic approach to be easily applied for different proteins or peptides even in matrices containing low molecular weight sulphur impurities.
The method can be used for the experimental determination of the extinction coefficient of a protein if combined with UV spectroscopy, but also as a reference (absolute quantification) or control method for in-house protein-based biological standard qualification. If needed, this method can be easily validated for specific protein products.