Isotopics™ – May 2018 | Edition 7

After way too many inches of snow over this past winter, we are ready for spring and summer here at CIL. To us, the warmer weather means that our favorite conferences are right around the corner and we look forward to debuting new products. If you have wanted to try one of our new products, but haven’t had a chance to order, there’s no better time than now.

Multiplexed Proteome Analysis with Neutron-encoded Stable Isotope Labeling in Cells and Mice

Overmyer, K.A.; Tyanova, S.; Hebert, A.S.; et al.

To help overcome the challenges of traditional metabolic labeling approaches in qualitative- and quantitative-based proteomic studies, Coon and colleagues developed an alternate labeling approach involving neutron-encoded (NeuCode™) lysine isotopologues. This approach exploits the minor mass differences in the neutron-binding energies of different isotopes of lysine for enhanced peptide identification and protein quantification under high resolution MS. This article details the sample preparation, processing, and analysis protocol required to effectively execute a 3-plex NeuCode experiment for relative protein quantitation of mouse models (SILAM) and cell lines (SILAC) by bottom-up LC-MS/MS (data-dependent acquisition). The application to alternate acquisition strategies and capability for higher multiplexing powers is also discussed.

A Mass Spectrometry-based Proteomic Analysis of Homer2-interacting Proteins in the Mouse Brain

Goulding, S.P.; Szumlinski, K.K.; Contet, C.; et al.

Scaffolding proteins of the Homer family are recognized as important indicators of several psychiatric disorders. To better understand their molecular function toward improved therapeutic strategies, the interactions of Homer proteins must be identified. Here, Goulding and colleagues employed co-immunoprecipitation (via rabbit polyclonal anti-Homer2 antibody) with nano-LC-MS/MS (DDA and SRM acquisition for discovery and verification, respectively) to identify interacting Homer2 proteins in mouse brain tissue (C57BL/6 strain). To help determine retention times, peptides from ¹⁵N Apolipoprotein A-I were used as iRT standards. Overall, the verification experiments revealed 18 Homer2-interacting proteins of high confidence, with 15 of those being novel interactors stemming largely from the N-methyl-D-aspartate receptor (NMDAR) signaling pathway (e.g., GluN1 and GluN2B). The results help demonstrate the functional implication of Homer2 in signal transduction.

Two Complementary Reversed-phase Separations for Comprehensive Coverage of the Semipolar and Nonpolar Metabolome

Naser, F.J.; Mahieu, N.G.; Wang, L.; et al.

Described here by Patti and colleagues is an improved LC-MS strategy for untargeted profiling of semi-polar (logP -2 to 1.5) and non-polar (logP 1.5 to 35) metabolites present in the neurotransmitter and lipid metabolism pathways. In their evaluations, method benchmarking was facilitated by authentic standards and E. coli cell extracts used together with the credentialing algorithm. The results revealed a tandem RP-LC approach (CORTECS T3 and C8 columns) to be superior for moderate-to-low polarity metabolites (e.g., phospholipids, triacylglycerols, nucleotides, nucleobases) relative to reference methods often used in metabolomics, delivering a 5-fold increase in the credentialed coverage over this molecular range. Complementing this approach with HILIC-MS extended the breadth for comprehensive metabolite profiling.

METLIN: A Technology Platform for Identifying Knowns and Unknowns

Guijas, C.; Montenegro-Burke, J.R.; Domingo-Almenara, X.; et al.

METLIN is a comprehensive metabolite database designed for metabolomic MS/MS analysis. This repository was developed initially in 2003 to help researchers identify known metabolites in their untargeted MS research. Through the use of MS/MS data (empirical and in silico generation aided by ¹³C-labeled yeast extract) and advanced informatic tools (e.g., “Neutral Loss and Fragment Similarity Search”), METLIN can now effectively characterize unknowns as well. The advancements are described and exemplified herein. Taken together, this should help facilitate new application opportunities in systems biology studies.

Noninvasive Liquid Diet Delivery of Stable Isotopes into Mouse Models for Deep Metabolic Network Tracing

Sun, R.C.; Fan, T.W.; Deng, P.; et al. 

To help overcome the challenges associated with in vivo tracer studies of model animals, Fan and colleagues developed a liquid diet procedure to deliver ¹³C₆-glucose into NSG mice. The administration is effective and noninvasive, enabling extensive metabolic tracing of a myriad of biochemical pathways, as quantified in tissue-based MS and NMR analysis. This developed approach was ultimately applied to patient-derived lung tumor xenografts as a function of mouse generations to track tumor-specific metabolic networks in NSG mice and compared to other tissue culture models to probe the effects of cancer cell metabolism on the tumor microenvironment.

A Timeline of Stable Isotopes and Mass Spectrometry in the Life Sciences

Lehmann, W.

Here, Wolf-Dieter Lehmann chronicles the development of stable isotopes (principally ¹³C, D, ¹⁵N, and ¹⁸O) and the MS-based techniques used for analysis. The review is divided into time segments (1920-1950, 1950-1980, 1980-2000, and 2000-2010), with methods and applications discussed therein. Included in the highlights is the use of stable isotopes (as quantitative standards or metabolic tracers) with LC- or GC-MS to address questions in ‘omics (e.g., metabolomics, proteomics, and lipidomics), metabolism, and forensics, among other fields of application.