The practice of quantifying proteins by peptide fragments from enzymatic proteolysis (digestion) was assessed regarding accuracy, reliability, and uncertainty of the results attainable. Purified recombinant growth hormone (rhGH, 22 kDa isoform) was used as a model analyte. Two tryptic peptides from hGH, T6 and T12, were chosen to determine the amount of the protein in the original sample. Reference solutions of T6 and T12 (isotopically labeled forms), value assigned by quantitative amino acid analysis (AAA) after complete hydrolysis, were used as internal standards. The accuracy of protein quantification by fragments T6 and T12 was evaluated by comparison of peptide results to those obtained for the same rhGH sample by AAA. The rate of cleavage (and thus the experimental protocol used) turned out to be crucial to the quality of results in protein quantification using enzymatic fragments. Applying a protocol customarily found in (qualitative) bottom-up proteomics gave results significantly higher than the target value from AAA (+11% with T6 and +6% with T12). In contrast, using a modified protocol optimized for fast and complete hydrolysis, results were unbiased within the limits of uncertainty, while the time needed for completion of proteolysis was considerably reduced (30 min as compared to 1080-1200 min). The method assessed highlighted three important criteria deemed necessary for successful protein quantification using proteolysis-based mass spectrometry methods. These are the following: the requirement for both the selected peptides and labeled internal standard to be stable throughout digestion; the correct purity assignment to the selected peptide standards; the proof of equimolar release of the selected peptides. The combined (overall) uncertainty for protein quantification was established by combination of estimates obtained for individual components and found to be U ) 4% for this example. This uncertainty is of the same order as that typically attainable in quantification of "small" organic molecules using liquid chromatography/isotope dilution mass spectrometry.The past decade has seen a significant increase in the development of mass spectrometric methods for protein quantification.1,2 Although it is viable to directly analyze intact proteins 3,4 by liquid chromatography/mass spectrometry (LC/MS), most of the examples of quantification that have been described are based on specific cleavage by enzymatic proteolysis (digestion) of the proteins down to smaller fragments, most of which are still long enough in amino acid sequence to provide specificity for the precursor protein, even in a complex mixture. [5][6][7][8][9][10][11][12][13][14] This enables the simplification of the quantification process to the analysis of short sequences of amino acids which are amenable to standard LC/MS techniques. Using isotopically labeled forms of the peptides as internal standards potentially introduces the advantages of reliability, accuracy, and repeatability into protein quantification that have been demonstrat...

BACKGROUND: Standardization of hemoglobin (Hb)A 1c measurements is a process of considerable interest for quality assurance in diabetes management. To contribute to continuous quality improvement and fulfillment of the requirements for reference measurement procedures according to the standards of the International Organization for Standardization, we developed a calibration system of highest metrological order using isotope dilution-mass spectrometry with a reference material.

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Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115, was coordinated by the Bureau International des Poids et Mesures (BIPM) and the Chinese National Institute of Metrology (NIM). Eight Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of human C-peptide (hCP) present as the main component in the comparison sample for CCQM-K115. The comparison samples were prepared from synthetic human hCP purchased from a commercial supplier and used as provided without further treatment or purification. hCP was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of short (up to 5 kDa), non-cross-linked synthetic peptides/proteins. It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used a peptide impurity corrected amino acid analysis (PICAA) approach as the amount of material that has been provided to each participant (25 mg) is insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, both the BIPM and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the hCP mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, mass balance or any other approach to determine the hCP purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the hCP mass fraction. In particular it allows participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification. More detailed studies on the identification/quantification of peptide related impurities and the hydrolysis efficiency revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the hCP mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that only the most exhaustive and elaborate set of results is taken for the calculation of the KCRVPepImp. The KCRVPepImp for the peptide related impurity mass fractions of the material was 83.3 mg/g with a combined standard uncertainty of 1.5 mg/g. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide related impurity profile indicates that in many cases only a very small number of impurities have been identified and quantified resulting in an underestimation of the peptide related impurity mass fractions. The approach to obtain a KCRVhCP for the mass fraction of hCP is based on a mass balance calculation that takes into account the most exhaustive and elaborate set of results for the peptide related impurities KCRVPepImp, the TFA mass fraction value, water and other minor counter ions obtained by the coordinating laboratories. Differences in the quality of the results obtained for both peptides related impurity mass fractions and hCP mass fractions are better weighted and reflected in smaller uncertainties. The KCRVhCP for CCQM-K115 is 801.8 mg/g with a corresponding combined standard uncertainty of 3.1 mg/g. In general, mass balance approaches show smaller uncertainties than PICAA approaches and the majority of results obtained by the PICAA approach are in agreement because of larger corresponding uncertainties. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).