Measurement of protein in vivo turnover rate with metabolic labeling using LC-MS

Understanding the protein dynamics of a drug target is important for pharmaceutical research because it provides insight into drug design, target engagement, pharmacodynamics and drug efficacy. Nonradioactive isotope labeling has been the method of choice for protein turnover measurement thanks to the advancement of high-resolution mass spectrometry. While the changes in proteome in cell cultures can be monitored precisely, as the culture media can be completely replaced with ² H-, ¹⁵ N- or ¹³ C-labeled essential amino acids, quantifying rates of protein synthesis in vivo is more challenging. The amount of isotope tracer that can be administered into the body is relatively small compared with the existing protein, thus requiring more sensitive detection, and the precursor-product labeling relationship is more complicated to interpret. The purpose of this review is to provide an overview of the principles of in vivo protein turnover studies using deuterium water (² H₂ O) with an emphasis on targeted protein analysis by hybrid LC-MS assay platforms. The pursuit of these opportunities will facilitate drug discovery and research in preclinical and clinical stages.