Fully traceable absolute protein quantification of somatropin that allows independent comparison of somatropin standards.

BACKGROUND: Measurement traceability in clinical chemistry is required to standardize clinical results irrespective of the measurement procedure and laboratory. The traceability of many protein substances is maintained by reference to the first standard produced, which may no longer exist, with values assigned by consensus. Independent methods that provide traceability to the Système d'Unité International for all relevant properties of a protein standard could remove reliance on the original standard preparations. METHODS: We developed a method based on the traceable quantification of tryptic peptides released from the protein by isotope dilution mass spectrometry to compare 2 standard preparations of somatropin (recombinant human growth hormone), WHO 98/574 and Ph.Eur.CRS S0947000. Relative quantification using isotope-coded affinity tagging, isobaric tagging for relative and absolute quantification, and standard additions were also performed to validate the digestion method used and to determine whether any modifications were present. RESULTS: The total somatropin content in both materials was determined and an uncertainty estimation undertaken [WHO 2.19 +/- 0.21) mg/vial, European Pharmacopeia 2.06 +/- 0.21 mg/vial]. Each uncertainty in this paper is a fully estimated uncertainty, with 95% CI (k = 2). Isotope coded affinity tag and standard addition results fully validated the robustness of the digestion method used. In addition, iTRAQ (isobaric tagging for relative and absolute quantification analysis) identified 2 modifications, neither of which impacted the quantification. CONCLUSIONS: An independent method that does not rely on a preexisting protein standard has been developed and validated for the traceable value-assignment of total somatropin. The methods reported here address the amount of substance (mass fraction) of the standard materials but address neither biological activity nor other characteristics that may be important in assessing suitability for use as a calibrator.

Toward Système International d'Unité-traceable protein quantification: from amino acids to proteins.

Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To provide full SI traceability to measurements of protein concentration we demonstrated a method of SI traceable peptide quantification in which the peptide standards used were quantified by an amino acid analysis method that incorporated double exact matching IDMS and amino acid standards of known purity. The concentration of the protein was therefore determined based upon the concentration of tryptic peptides, which in turn had been quantified based upon amino acid standards. This allowed fully SI-traceable measurements of protein concentration to be made. Important caveats in the implementation of this approach are also discussed and examples of how these can have detrimental effects on the measurements are shown.

Principles of Fourier transform ion cyclotron resonance mass spectrometry and its application in structural biology.

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has become increasingly significant within recent years. The inherently ultra-high resolution and mass accuracy allow unequivocal assignments of chemical formulae to be made and further structural elucidation can be conducted through the utilization of tandem mass spectrometry techniques. With the advent of electrospray ionization (ESI), FT-ICR mass spectrometry has become a powerful tool for the investigation of biological macromolecules, such as the study of non-covalent interactions of proteins. In this article, the basic principles are highlighted, some of the techniques employed are described and examples of applications are provided, with particular respect being paid to the field of characterization of biomolecules.