Isotopics – July 2016 | Edition 3

We are delighted to present the third edition of our IsoTopics™ newsletter! In this issue, we feature several articles by researchers who have further advanced proteomics, metabolomics, and clinical research. Some of these researchers have used traditional CIL products in new, exciting ways and some have used brand-new CIL products to solve a problem that may not have been possible in the past. We hope that these articles inspire further development in mass spec applications and collaboration among researchers from various disciplines.

Determining Synthesis Rates of Individual Proteins in Zebrafish Danio rerio with Low Levels of a Stable Isotope Labelled Amino Acid

Geary, B.; Magee, K.; Cash, P.; et al.

The zebrafish (Danio rerio) is an established model organism for studying cardiovascular disease. To better understand protein flux in the myocardium, Doherty MK et al. fed zebrafish low levels of L-Leu (d7), then measured its synthesis and turnover rates over an eight-week labeling period. Bottom-up LC-MS/MS analysis of the heart tissue digests revealed rates of protein synthesis and incorporation for 661 proteins. These were predominantly intracellular, involved in metabolic processes and enriched in nine biochemical pathways (including glycolysis, ATP synthesis, and blood coagulation). This study marks an important step toward integrating genotype-phenotype data.

NeuCode Proteomics and Bap1 Regulation of Metabolism

Baughman, J.M.; Rose, C.M.; Kolumam, G.; et al.

Capitalizing on the merits of neutron encoding (NeuCode), Coon J et al. applied this metabolic-labeling technique to genetically engineered mice models to investigate mammalian biology in vivo. After preliminary experiments on the labeling efficiency and quantitative accuracy, three tissues (namely liver, pancreas, and spleen) were selected for further evaluation of Bap1, a deubiquitinating enzyme with cancer association. This research highlighted the utility of 4-plex NeuCode in mammals and revealed Bap1 to be a key regulator of metabolic homeostasis.

Stable Isotope Labeling of Algal Biomass Carbohydrates

McConnell, B.O.; Antoniewicz, M.R.

Given the growing interest in algal biomass in biological and biofuel applications, Antoniewicz M et al. developed a new analytical method to more conveniently quantify carbohydrates in C. vulgaris. The method involved two-step HCl hydrolysis, chemical derivatization of monomer sugars by aldonitrile propionate, and GC-MS analysis. The use of ¹³C-labeled carbohydrates (e.g., glucose) enabled their composition and metabolic flux to be determined in C. vulgaris media grown autotrophically and heterotrophically.

Biochemical Pathways Affected by Mitochondrial Stress

Bao, X.R.; Ong, S.E.; Goldberger O.; et al. 

In a landmark study, Mootha VK et al. integrated metabolomic, proteomic, and RNA profiling strategies to identify hypotheses of the biochemical pathways affected by mitochondrial stress. The results indicated a link between mitochondrial dysfunction and serine/homocysteine metabolism. Further examination of Ser biosynthesis and transsulfuration revealed mechanisms by which mitochondrial dysfunction might compromise one-carbon synthesis in mammalian cells.

MS-based Quantitative Plasma Proteomics

Percy, A.J.; Byrns, S.; Pennington, S.R.; et al.

This review article provides a retrospective and prospective five-year view of quantifying human plasma proteins for biomarker development in a clinical setting. The highlighted methods are categorized under a fit-for-purpose approach (i.e., from Tier 1 to Tier 3 for clinical and exploratory assays, respectively) and primarily utilize a bottom-up methodology with stable isotope-labeled standards (at the protein or peptide level) for quantification. Discussed are the challenges, requirements, and strategies for translating candidate protein biomarkers from research to the clinic.