Development of Proteomics-Based Methods for Protein Quantification

NIST is developing methods for the quantification of clinically-relevant protein biomarkers by mass spectrometry. Some methods are intended to establish reference measurement procedures for the production of standard reference materials, which establish traceability and improve the accuracy of routine clinical measurements by standardizing the calibration procedures. Such traceability is required by the European Directive on in vitro diagnostics (IVD) for all measurements in laboratory medicine. Other quantitative mass spectrometry based methods will be used to characterize and improve routine clinical immunoassays for protein biomarkers. NIST is also working to demonstrate the reproducibility of quantitative mass spectrometry methods through participation in inter-laboratory studies.

Reference measurement procedures and standard reference materials help ensure that routine clinical assays provide accurate results across different laboratories and over time. Lists of such higher-order methods are published by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). While there are many reference measurement procedures for small-molecule organic and inorganic species, few exist for clinically relevant proteins. In order to meet this need, new measurement procedures must be developed. As the field of proteomics matures, it is likely that more protein biomarkers will be discovered and used for clinical diagnoses. These potential biomarkers can be verified through quantitative mass spectrometry methods. Existing clinical immunoassays for protein biomarkers already in use can be improved by establishing traceability to more accurate mass spectrometry methods.

A “bottom-up” proteomics approach is used to quantify clinically relevant proteins in serum/plasma. Specifically, peptides are generated through the enzymatic digestion of the target protein in the sample. Because the analyte (protein) and measurand (peptides) are different, care must be taken to account for sources of bias in the enzymatic digestion process that converts the analyte to the measurand. This is accomplished through the use of appropriate calibration curves and ideally the use of isotopically labeled proteins as internal standards. These labeled proteins are expressed in E. coli or yeast with media containing nitrogen-15 labeled ammonium chloride as the only nitrogen source. Further measurements can establish the final percent incorporation of nitrogen-15 in the recombinant protein, and ensure the labeled and unlabeled forms have similar antibody binding and digestion characteristics. Affinity purification techniques based on magnetic-bead bound monoclonal antibodies capture the labeled and unlabeled proteins in the clinical matrix. Following separation from the matrix and release from the antibody, the proteins are digested and the peptides are quantified using liquid chromatography coupled to multiple-reaction-monitoring mass spectrometry.

NIST also developed study materials for a National Cancer Institute, Clinical Proteomics Technology Assessment for Cancer (CPTAC), inter-laboratory evaluation of the ability of mass spectrometry methods to simultaneously quantify multiple target proteins in serum/plasma. This study established the reproducibility of quantitative mass spectrometry methods and the appropriateness of this technology for protein biomarker verification.

Major Accomplishments:

• Affinity purification approaches for troponin and c-reactive protein have been developed.
• Quantitative mass spectrometry methods for troponin (using labeled signature peptides) and c-reactive protein (using labeled c-reactive protein) have been developed.
• The CPTAC protein quantification inter-laboratory study was completed.