Isotopics – November 2016 | Edition 4
We are excited to present our latest edition of IsoTopics™! It has been a year since the first publication of our newsletter, and we sincerely appreciate your interest and comments over the past 12 months. We hope you have found the content useful and informative. Please continue to send us feedback as we continually strive for improvement. As with past issues, this edition features several articles utilizing stable isotopes to initiate further discussion and interest within the MS community.
Multiplexed MRM-based Assays for the Quantitation of Proteins in Mouse Plasma and Heart Tissue
Percy, A.J.; Michaud, S.A.; Jardim, A.; et al.
Described here is the rigorous development and application of highly multiplexed assays for the in-depth quantitation of hundreds of proteins in mouse plasma and heart tissue samples. The method features a targeted MRM approach with stable isotope-labeled peptides used as internal standards. The validated assays have disease relevance and association to biological/molecular processes, and are suitable for molecular phenotyping as well as biomarker assessment studies. A collection of the validated plasma assays have been assembled into a quality control (QC) and a biomarker assessment kit (BAK) for the research community. The PeptiQuant™ BAK-81, for example, enables 81 proteins spanning a four order of magnitude concentration range to be quantified in mouse plasma samples through bottom-up LC-MRM/MS and a reverse standard curve approach.
Optimal Tracers for Parallel Labeling Experiments and 13C Metabolic Flux Analysis: A New Precision and Synergy Scoring System
Crown, S.B.; Long, C.P.; Antoniewicz, M.R.
Antoniewicz MR and colleagues have developed a new scoring scheme to identify optimal 13C tracers in single and parallel labeling experiments of metabolic flux analysis (MFA) studies. The scheme is based on precision and synergistic metrics that, for the first time, accounts for nonlinear flux intervals and avoids biases due to flux value normalization. The metrics were evaluated against hundreds of labeling schemes through extensive in silico simulations and in vivo labeling experiments of E. coli measured by GC-MS. Matched findings revealed the double-labeled glucose tracers (1,6-13C) and (1,2-13C) to be optimal for MFA of E. coli.
N-linked Glycosite Profiling and Use of Skyline as a Platform for Characterization and Relative Quantification of Glycans in Differentiating Xylem of Populus trichocarpa
Loziuk, P.L.; Hecht, E.S.; Muddiman, D.C.
Given the importance of glycomic characterization in biological systems, Muddiman DC and co-workers developed a new workflow for the combined profiling of deamidated glycosites and N-linked glycans in the differentiating xylem of Populus trichocarpa. The workflow involved a FANGS-INLIGHT coupled methodology, which featured hydrazide tagging (via the heavy/light INLIGHT reagent) of the enzymatically released N-linked glycans and bottom-up preparation of the deamidated glycoproteins from the filter, prior to LC-MS analysis. This protocol enabled 27 N-glycans and 502 glycosylated proteins to be identified, several of which have potential linkages to lignin biosynthesis. In addition, Skyline was demonstrated to accurately and precisely quantify N-glycans; thus, providing a new software solution for automated glycan analysis.
Determining the Turnover of Glycosphingolipid Species by Stable-Isotope Tracer Lipidomics
Skotland, T.; Ekroos, K.; Kavaliauskiene, S.; et al.
In an effort to better understand the synthesis and turnover rates of glycosphingolipid (GSL) species, Skotland T et al. conducted a time-course study with HEp-2 cells grown in the absence/presence of a stable isotope-labeled substrate (either U-13C glucose or serine). The de novo biosynthesis/degradation of unlabeled/labeled ceramide and GSLs were evaluated by LC-MRM/MS. Found was a class-dependent correlation with the formation and turnover of sphingolipids and GSLs according to the labeled substrate incorporated. This enabled pathway-specific measurements to be made.
Quality Management in Clinical Application of Mass Spectrometry Measurement Systems
Vogeser, M.; Seger, C.
Given the increasing application of quantitative MS methods (with stable isotope internal standardization) to clinical samples, aspects of quality and risk management must be considered for the assays to ultimately be fit for diagnostic purposes. These aspects are extensively weighed in Vogeser and Segers recent review article. Addressed therein are pre-implementation strategies, quality assurance tools, validation approaches, assay assessment, and regulatory practices.