Abstract
Mass spectrometry is a useful method for the measurement and comparison of protein turnover. Classical methods measure the incorporation of stable isotope labeled amino acid tracers into abundant proteins via GC-MS. Here we present results from two complimentary approaches that measure isotope enrichment in tryptic peptides of target proteins. The approaches utilized a triple stage quadrupole mass spectrometer and (1) stable isotope labeled amino acid tracers or (2) heavy-oxygen water tracers. In humans, we compared the fractional synthetic rate values calculated using D3-labled leucine enrichment data obtained with the classical GC-MS amino acid method and the peptide method. In mice, we measured ApoB100 protein turnover in plasma-isolated LDL and VLDL using heavy-oxygen water tracers. The selectivity of both approaches is based on the amino acid sequence of tryptic peptides and thus is not restricted to the analysis of high abundance proteins that can be isolated by 1D SDS PAGE. This lecture will describe the fundamental and practical considerations for each method and highlight the differences in how these assays were developed and applied.