Additional Technical Details:
A “bottom-up” proteomics approach is being used to quantify clinically relevant proteins in serum/plasma. Specifically, peptides are generated through the enzymatic digestion of the target protein in the sample. Quantitation can then be performed by adding known amounts of isotopically labeled peptides as internal standards. This technique has been utilized to quantify troponin. However, this approach is not ideal because it may fail to compensate for incomplete digestion of the proteins or other variables in sample processing. Therefore we have investigated the production of full-length stable isotope-labeled proteins that can be used as internal standards and spiked into the sample at the beginning of the analysis. These labeled proteins are expressed in E.coli or yeast with media containing nitrogen-15 labeled ammonium chloride as the only nitrogen source. To date, an 15N-labeled form of C-reactive protein (CRP) has been produced and is currently being evaluated for measurement of CRP in serum.
Many proteins of clinical interest are present in low abundance relative to other serum proteins. As a result, direct quantitation of these proteins at normal levels in serum by mass spectrometry is problematic. Affinity purification techniques based on magnetic-bead bound monoclonal antibodies can capture both labeled and unlabeled proteins in clinical matrices. 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. This mechanism has been used to isolate CRP from a serum matrix, and preliminary results indicate that affinity purification combined with mass spectrometry can be used to quantify CRP at physiologically relevant levels. This work represents the first step toward developing robust higher-order methods for protein quantitation, and toward the development of natural matrix reference materials for these analytes.
Start Date:October 1, 2000
Lead Organizational Unit:mml
David M. Bunk
Related Programs and Projects:
Metallomics: A Multidisciplinary Approach for the Qualitative and Quantitative Determination of Metalloproteins Used as Health and Disease Markers
Bunk, D.M., “Reference Materials and Reference Measurement Procedures: An Overview from a National Metrology Institute,” Clin. Biochem. Rev., 28(4):131-137 (2007).
Arsene, C.G., Ohlendorf, R., Burkitt, W., Pritchard, C., Henrion, A., O’Conner, G., Bunk, D.M., and Güttler, B., “Protein Quantification by Isotope Dilution Mass Spectrometry of Proteolytic Fragments: Cleavage Rate and Accuracy,” Anal. Chem., 80(11):4154-4160 (2008).
Panteghini, M., Bunk, D.M., Christenson, R.H., Katrukha, A., Porter, R.A., Schimmel, H., Wang, L., Tate, J.R., and the IFCC Working Group on Standardization of Troponin I, “Standardization of Troponin I Measurements: An Update,” Clin. Chem. Lab. Med., 46(11):1501-1506 (2008).