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.
- 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.