Isotopics – April 2017 | Edition 5
We are delighted to present the following newsletter for you to take along on your “spring break” this year. Even if you’re surrounded by mass specs instead of palm trees, we think you’ll enjoy the featured articles.
SWATH-based Proteomics Identified Carbonic Anhydrase 2 as a Potential Diagnosis Biomarker for Nasopharyngeal Carcinoma
Luo, Y.; Mok, T.S.; Lin, X.; et al.
Given the prevalence of nasopharyngeal carcinoma (NPC) in Asian populations, there is an increasing need for diagnostic protein biomarkers. To that end, Luo and colleagues used a SILAC-labeled spike-in standard (consisting of 13C6/15N2-Lys and 13C6/15N4-Arg) for a super-SILAC shotgun MS analysis of human nasopharyngeal tissue samples. Through SWATH (i.e., sequential window acquisition of all theoretical fragment ion spectra)-MS analysis, 1,414 proteins were identified and quantified, with a subset of 29 statistically found to be differentially expressed. Verification with MS and immunoblotting data revealed carbonic anhydrase 2 to be a novel, potential biomarker worthy of future exploration.
Serotonin Biosynthesis as a Predictive Marker of Serotonin Pharmacodynamics and Disease-Induced Dysregulation
Welford, R.W.; Vercauteren, M.; Trébaul, A., et al.
In a metabolomics-based tracer study, Welford and colleagues used 13C-labeled tryptophan as an oral tracer to measure/quantify the biosynthesis and metabolism of serotonin in rats. Here, the flux and pharmacokinetics of serotonin pathway metabolites were measured in various tissues by LC-MRM/MS. The pharmacodynamic effects of serotonin synthesis inhibition in vivo were also investigated. This study should help facilitate the development of improved serotonin synthesis inhibitors, which could have therapeutic potential in diseases such as lung fibrosis, osteoporosis, and obesity.
Historical and Contemporary Stable Isotope Tracer Approaches to Studying Mammalian Protein Metabolism
Wilkinson, D.J.
A formative article by Daniel Wilkinson of the University of Nottingham provided a historical perspective on the evolution of stable isotope tracers in relation to MS-based technological advancements. The impetus of the highlighted studies was to investigate in vivo mammalian metabolism (e.g., protein, lipid), either at a whole body or analyte-specific level. The future of such research is broad, with its potential impact in clinical arenas and biomarker diagnostics additionally discussed in the context of MS ’omics research.
Comparative Analysis of INLIGHT™-labeled Enzymatically Depolymerized Heparin by Reverse-phase Chromatography and High-performance Mass Spectrometry
Mangrum, J.B.; Mehta, A.Y.; Alabbas, A.B., et al.
In an effort to overcome the analytical and technical challenges associated with glycosaminoglycan (GAG) analysis, Mangrum and colleagues applied the INLIGHT™ (individuality normalization when labeling with glycan hydrazide tags) labeling strategy to characterize and quantitatively analyze heparin digests (digHep). Experimentally, the digHep samples were independently tagged with a light (12C6) or heavy (13C6) hydrazide reagent (phenyl 2-GPN) before equimolar mixing and comparative analysis by reversed-phase LC-MS and -MS/MS.
13C Metabolic Flux Analysis of Acetate Conversion to Lipids by Yarrowia lipolytica
Liu, N.; Qiao, K.; Stephanopoulos, G.
To better understand the metabolism of yeast Yarrowia lipolyticain the presence of volatile fatty acids, Stephanopoulos and colleagues performed a stationary 13C metabolic flux analysis using two isotopically labeled acetate tracers (13C and 13C2) as the feedstock. Distribution maps were constructed from empirical (by GC- and LC-MS) and theoretical observations of two different yeast extracts: MTYL037 (served as the control) and MTYL065 (engineered lipid overproducer). Lipid synthesis pathways investigated during the growth and production phases included the TCA, glyoxylate shunt, gluconeogenesis, and pentose phosphate. The results indicated a flux dependence on strain and phase, with malate transport and pyruvate kinase playing critical roles in energy production.