Dynamic Proteomics

Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry

“…Following the administration of 2 H 2 O into the body water pool in vivo or into cell culture media in vitro, 2 H atoms become stably incorporated via enzyme catalyzed reactions into C-H bonds of nonessential amino acids, glycerol-3-phosphate, fatty acids, cholesterol, hexoses and pentoses (ribose and deoxyribose). Subsequent analysis of 2 H labelling of these constituents in macromolecules provides a highly sensitive measure of the rates of synthesis/turnover of lipids [15,16,17,18,19,20,21,22,23,24,25,26,27], proteins [15,28,29,30,31,32,33,34], DNA [15,35,36], RNA [15] and glucose/glycogen [25,26,37,38,39,40,41,42,43] and an objective assessment of fundamental cellular processes that include, but are not limited to, rates of cell and organelle proliferation, cell and tissue biomass turnover, de novo lipogenesis and gluconeogenesis. Such measurements have tremendous application to researchers in the field of cancer, immunology, cardiovascular disease, metabolism, diabetes, obesity, developmental biology, as well as the biotechnology and agricultural sectors.…”

Exploring ribosome composition and newly synthesized proteins through proteomics and potential biomedical applications

“…For example, for quantification of Drosophila (fruit fly) proteomes [45], for preparation of 15 N-labeled protein standards from mice to compare unlabeled proteomes [46], for identification of variation in expression and turnover of dihydropyrimidinase-related proteins DPYSL3 and DPYSL5 during brain development by incorporation of 15 N into mice diet [47], and for determination of protein turnover rates on whole proteome scale by restricting all the nitrogen in the rodent diet to 15 N (spirulina algae) with reversion to normal 14 N diet with time points at days to years [48]. In addition, there have been various reviews published discussing both the strengths and the weaknesses of chemical and metabolic labeling of model organisms [49], the merits and difficulties of labeling methods for assessing protein turnover and dynamics [50–51], and a review article of stable isotopes and mass spectrometry in life sciences covering a time period from 1920 to 2016 [52]. Since SILAC and non-canonical amino acids labeling are discussed later in more details as the techniques of proteomic workflow used for metabolic labeling of newly synthesized proteins and their enrichment, identification and quantitation in the cells, tissues or whole animals, the brief basic description of each technique is included in following sections.…”

Strategies for Extending Metabolomics Studies with Stable Isotope Labelling and Fluxomics

“…and observing their metabolic incorporation and/or dilution in biological systems, it is also possible to use other labelling approaches combined with mass spectrometric and/or NMR analysis to understand cellular metabolism and growth characteristics. For example, the use of deuterated “heavy” water ( 2 H 2 O) has proven to be a particularly powerful approach for measuring the synthesis rates of proteins [71,72,73,74,75], lipids ( de novo lipogenesis, esterification, and chain elongation) [67,76,77,78], DNA [79,80], and RNA [81], thus providing information on biomass synthesis and cell division rates, both in vivo and in vitro . Furthermore, 2 H 2 O labelling can be used to quantify the pathways used to produce glucose and glycogen, thus providing a way to measure gluconeogenic and glycogenolytic/glycogenic pathway activity [82,83,84,85,86,87].…”

“…The toxic effects can be largely avoided by maintaining 2 H 2 O enrichments of body water/cell culture media to <10%, although the kinetic isotope effect should be considered when modelling and interpreting data from 2 H 2 O labelling [92,93]. 2 H 2 O has also recently been combined with other “omics” technologies including proteomics [74,75,94,95,96], permitting the determination of synthetic rates of individual proteins on a global level in vivo , and imaging mass spectrometry [97], permitting the imaging of newly synthesized lipids in tissue histology samples, termed “kinetic histochemistry”. Thus, in addition to using metabolomics and substrate specific tracers for mapping metabolic pathway activity, 2 H 2 O labelling can provide complementary information on the synthetic rates of numerous cellular components and their biosynthetic pathways (i.e., gluconeogenesis vs. glycogenolysis; de novo lipogenesis vs. preformed fatty acid esterification).…”