Measurement of intact proteins by mass spectrometry can be problematic. In order to make proteins more amenable to mass spectrometric detection, the proteins are commonly digested with a proteolytic enzyme prior to analysis. Quantitation can then be performed by adding known amounts of isotopically labeled peptides as internal standards. However, this approach is not ideal because it may fail to compensate for incomplete digestion of the proteins or other variables in sample processing. A full-length stable isotope-labeled protein that could be spiked into the sample at the beginning of the analysis would account for the multiple variables that may affect the final levels of the measured peptides. Implementing this approach represents a key step in developing robust higher-order methods for protein quantitation.
Proteins are of significant interest as potential indicators (biomarkers) of health or disease. Clinical methods employed to measure these analytes are often based on immunological affinity or enzyme activity. In order for such tests to be traceable to standards of a higher order, reference methods and reference materials must be established for clinically relevant proteins. Methods based upon liquid chromatography - mass spectrometry (LC-MS/MS) are well suited to this purpose. However, the lack of isotopically labeled proteins that can serve as internal standards for these measurements has hindered progress in this area. Therefore, NIST has been evaluating expression systems to produce isotopically labeled forms of proteins of clinical diagnostic interest.
Additional Technical Details
Initial work has focused on expression of a stable isotope-labeled form of C-reactive protein (CRP). This protein is produced in the body in response to inflammation, and measurement of CRP levels can provide evidence of disease risk or progression. At the present time, there are no reference materials with CRP concentrations in the normal to moderately elevated range, primarily because of a lack of higher order methods for value assignment. Recombinant C-reactive protein (rCRP) was expressed in E. coli with media containing 15N-ammonium chloride as the sole nitrogen source. The resulting 15N-labeled rCRP (15N-rCRP) was purified sequentially by immobilized metal affinity and phosphatidylcholine binding with a yield of approximately 60 micrograms. The protein purity of 15N-rCRP was greater than 99% as assessed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Incorporation as a percent of total nitrogen was estimated to be greater than 99% as determined by isotopic profile analysis. Proteolytic digestion of mixtures of CRP and the isotopically labeled CRP indicated that they behaved in a similar manner. The labeled protein is now being investigated as an internal standard for quantitation of CRP in serum. The knowledge gained from the rCRP will be beneficial in establishing expression and purification systems for other isotopically proteins of interest.
- An intact stable isotope-labeled protein has been expressed with a high level of 15N incorporation.
- The isotopically labeled protein retains the structural features of the native protein and is anticipated to be suitable as an internal standard.
David M. Bunk
Eric L. Kilpatrick
Karen W. Phinney
Illarion V. Turko
Development of Reference Methods and Reference Materials for Proteins
Metallomics: A Multidisciplinary Approach for the Qualitative and Quantitative Determination of Metalloproteins Used as Health and Disease Markers
Development of Proteomics-Based Methods for Protein Quantification
A Collaborative Network to Support Cancer Biomarker Discovery and Validation (NCI CPTAC)
Development of Reference Methods and Reference Materials for the Determination of Cardiac Troponin I in Human Serum
Development of a Reference Measurement Procedure for C-Reactive Protein
Development of an Amino Acid (in Solution) Standard Reference Material
Chromatographic Separation of Unfolded Proteins for Protein Quantification