Isotopics – April 2016 | Edition 2
We are delighted to present the second edition of our IsoTopics™ newsletter! In this issue, we feature several articles published in the past few months that have had a significant impact on the mass spec community and cover a wide range of applications for stable isotopes. We hope they inspire further development in the MS field and collaboration among researchers from various disciplines.
Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phosphosignaling
Kennedy, J.J.; Yan, P.; Zhao, L.; et al.
With the need for highly robust quantitative assays for phospho-signaling in mind, Paulovich, A. et al. developed an antibody-free, multiplex IMAC-MRM method with isotopically labeled peptide standards to quantify 107 phosphosites (93 proteins) in human cell lysates. These phosphopeptides are involved in DNA damage-response (DDR) signaling pathways and were quantified with excellent linearity (≥3 orders of magnitude) and a high degree of precision (<13% intra- and inter-assay CV). Opportunities to assess and expand this phosphopeptide panel are available for cell signaling quantitation through access to the online resources provided with this article.
Discovery of Serum Protein Biomarkers in the mdx Mouse Model and Cross-Species Comparison to Duchenne Muscular Dystrophy Patients
Hathout, Y.; Marathi, R.L.; Rayavarapu, S.; et al.
Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration. Since current markers for DMD have limited specificity, efforts to expand the protein biomarker panel through comprehensive MS profiling are being explored. In this article, a concordant panel of serological biomarkers was identified in dystrophin-deficient mouse models – the naturally occurring mdx-23 and genetically engineered mdx-52 – and DMD patients by SILAM and label-free LC-MS/MS. These protein markers are associated with dystrophin deficiency and age-related muscle pathology, paving the way for their potential use as a readout tool for dystrophinopathies.
Isotopic Ratio Outlier Analysis of the S. cerevisiae Metabolome Using Accurate Mass Gas Chromatography / Time-of-Flight Mass Spectrometry: A New Method for Discovery
Qiu, Y.; Moir, R.; Willis, I.; et al.
Analyte identification is a major bottleneck in metabolomics studies. The isotopic ratio outlier analysis (IROA) technique, which utilizes full metabolic 13C labeling to distinguish between two cell populations (i.e., experimental and control), has proven valuable in LC-MS analysis to discriminate between metabolites of biological and nonbiological (i.e., chemical artifacts and noise) origin. Here, Kurland, I. et al. demonstrates the application of the IROA labeling technology to GC-MS profiling (via electron impact and chemical ionization) of the yeast (S. cerevisiae) metabolome. Using accurate mass GC-MS data in combination with a newly developed algorithm for formula elucidation, the structure/identity of 126 yeast metabolites was characterized. This catalog represents a first step toward the creation of clean spectral libraries for improved metabolite identification of experimental samples.
Gluconeogenesis and Glycogenolysis Measurement Review
Chung, S.T.; Chacko, S.K.; Sunehag, A.L.; et al.
Measurements of gluconeogenesis have been pivotal in elucidating the physiologic regulation of blood glucose homeostasis, as well as understanding the roles of glycogenolysis and gluconeogenesis in the pathophysiology of glucose metabolism in such conditions as obesity, metabolic acidosis, hypoglycemia, and diabetes. However, there is no gold-standard measure for quantifying the contribution of gluconeogenesis to total glucose production, and there has been significant controversy about specific methodologies and the ultimate result obtained. This review attempts to provide a comprehensive overview of the in vivo methodologies used to measure gluconeogenesis and glycogenolysis in humans. It discusses the real and theoretical advantages and limitations of each method and highlights the benefits of newer methodologies, such as deuterium oxide, which are simpler, more affordable, and less invasive than older techniques.
Recommendations for Peptides Used In Mass Spectrometry Assays
Hoofnagle, A.N.; Whiteaker, J.R.; Carr, S.A.; et al.
Clinical laboratories are beginning to employ bottom-up MS-based methods with stable isotope-labeled standards for protein quantitation of biosamples, yet the procedures for development (from peptide selection to handling) and analysis are not well standardized. This report provides a compendium of guidelines and specifications from experts in the field for effectively using internal peptide standards to quantify endogenous peptides in complex proteolytic sample digests. The goal of the recommendations is to achieve precise, relative quantitation that can be harmonized across laboratories with high a degree of repeatability/reproducibility.
Proteomic Maps of Breast Cancer Subtypes
Tyanova, S.; Albrechtsen, R.; Kronqvist, P.; et al.
Using MS-based proteomics for systems-wide profiling (instead of genomics and transcriptomics), Geiger, T. et al. employed FFPE filter-aided sample preparation (FASP) in combination with a super-SILAC approach to quantify >10,000 proteins (from >157,000 sequence-unique peptides) in clinical breast cancer tumor samples. The functional networks within and between breast cancer subtypes were examined, while novel cancer regulators and subtype-specific biological processes were discovered. To support clinical translation, a computational workflow based on support vector machine (SVM)-based classification was developed for protein identification according to breast cancer subtype.