**3. Metrology in protein quantification**

### **3.1 Backgrounds**

Protein is a complex molecule composed of one or more amino acid chains and constitutes a large part of the body's cells, tissues, and organs. This building block of the body has been shown to be a potential biomarker of human health risk assessment. Over the years, therefore, substantial efforts have been made for the development of bioanalytical approaches for accurate quantification of proteins. Monitoring protein levels is important to understand the biological mechanism and regulation of targeted proteins. In clinical practice, such approaches for protein quantification are used to compare biological samples from healthy and diseased patients, providing more accurate diagnosis of human disease. Thus, technologies for accurate quantification of protein biomarkers has been a high priority for many public health related institutions.

Nevertheless, measurement traceability, the essential element of metrology, is hardly established at all in protein quantification. There are several bioassay-based or instrumental methods for protein quantification that are frequently used. All these methods have to be calibrated to produce accurate results, but are not supported with reference calibration materials of metrological grades. Therefore, those methods work at best for comparison of relative amounts. Certified reference materials of proteins to provide measurement traceability to the SI unit of mole is yet to be developed. Our analytical capability is not as advanced as to quantify a whole protein accurately. However, quantification of appropriately reduced forms such as amino acids or peptides using isotopic dilution mass spectrometry (IDMS) should be a feasible approach to quantify proteins in a metrological manner. Of course, such approaches should also be supported by certified reference materials of the reduced forms for accurate calibration. The technical issues in the reduction approaches combined with the use of IDMS are described below.
