Development of Reference Methods and Reference Materials for Proteins

A number of proteins show promise as potential markers of health or disease, and commercial assays are being developed for these proteins. For example, measurement of the protein cardiac troponin I in serum has become the “gold standard” for the clinical diagnosis of myocardial infarction (heart attack). The accuracy of these clinical diagnostic measurements is essential for reliable diagnosis and cost-effective treatment of disease. Because different clinical assay manufacturers employ different calibrators and antibodies with different specificities, these commercial analytical systems consequently give results that are not comparable. Reference measurement procedures and standard reference materials can play an important role in clinical measurement standardization.

Accuracy and comparability of clinical assays is essential for timely and accurate medical interventions. However, studies have shown that large variations in results can occur among routine clinical assays. A clinical comparison among 14 commercial assay platforms revealed more than 20-fold differences in cardiac troponin I values. While there are many reference measurement procedures for small-molecule organic and inorganic species, few exist for clinically relevant proteins. NIST is developing higher-order mass spectrometry-based methodology capable of quantifying proteins present at low concentrations in complex biological matrices such as plasma or serum. These methods can then be used to provide a metrologically sound basis for routine clinical assays for these proteins.

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.

Major Accomplishments:

• Affinity purification approaches for troponin and CRP have been developed. 
• An intact stable isotope-labeled protein has been expressed with a high level of ¹⁵N incorporation.     
• Quantitative mass spectrometry methods for troponin (using labeled signature peptides) and C-reactive protein (using labeled C-reactive protein) have been developed.