Isotopics – October 2017 | Edition 6

Now that fall is here, we look forward to cooler weather and sharing the latest MS developments with you. In case you missed any over the past few months, here are several articles featuring novel approaches we think you will enjoy.

HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification

Ma, Y.; McClatchy, D.B.; Barkallah, S.; et al.

The noncanonical amino acid azidohomoalanine (AHA) has proven effective at measuring the synthesis and turnover of newly synthesized proteins (NSPs) in various biological samples, such as mouse tissue. Described here by Yates et al. is a new metabolic labeling method – termed HILAQ (heavy isotope labeled azidohomoalanine quantification) – for enriching and quantifying NSPs in HEK293T and HT22 cell lines. Experimentally, cells are pulse labeled with light or heavy AHA then mixed (1:1 w/w) and processed by bottom-up LC-MS. Using peptide AHA enrichment as opposed to protein AHA enrichment (as in the QuaNCAT protocol) was found to yield enhanced NSP quantification for improved monitoring of protein dynamics.

Multiplexed MRM-based Protein Quantitation Using Two Different Stable Isotope-labeled Peptide Isotopologues for Calibration

LeBlanc, A.; Michaud, S.A.; Percy, A.J.; et al.

Toward enhanced depth and precision/accuracy of multiplexed protein quantitation, LeBlanc et al. developed a new calibration approach utilizing two variations of stable isotope-labeled tryptic peptides (referred to as SIS 1 and SIS 2). The SIS 1 peptides have a C-terminal ¹³C/¹⁵N-label, while SIS 2 has an internal ¹³C/¹⁵N-labeled residue in addition to a C-terminal label. Procedurally, external curves and QCs are prepared in a surrogate matrix using a constant amount of SIS 1 and a variable amount of SIS 2. This double-SIS-peptide approach overcomes matrix-induced interference from endogenous peptides and, compared to conventional quantitative approaches, provides superior figures of merit for improved method development and assay validation.

Anion-Exchange Chromatography Coupled to High Resolution Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-Targeted Metabolomics

Schwaiger, M.; Rampler, E.; Hermann, G.; et al.

To help overcome the analytical challenges of ion-pairing-MS and GC-MS, Schwaiger et al. developed an innovative analytical workflow involving fast-scale anion-exchange chromatography and high resolution MS for simultaneous targeted/non-targeted metabolomic analysis. The approach utilized a fully C-labeled yeast extract as an internal standard for absolute quantitation and metabolic profiling of cancer cells in a single analytical run. The classes of metabolites qualified/quantified included (but was not limited to) organic acids, nucleotides, and sugar phosphates. Excellent figures of merit were achieved in both analysis modes, with quantifications of a 45-metabolite panel spanning a four order of magnitude concentration range (5 nM to 50 µM). Overall, this integrative approach opens up new opportunities for central carbon metabolism interrogations.

Metabolite Spectral Accuracy on Orbitraps

Su, X.; Lu, W.; Rabinowitz, J.D.

Spectral accuracy is an important metric of mass spectrometer performance, reflecting the ability to accurately quantify labeled forms. Rabinowitz et al. identify potential systematic errors in spectral accuracy that occur in high resolution Orbitrap measurements. These are fixed with proper choice of mass scan window together with a novel isotope correction algorithm – AccuCor (open source). The theory and experimental utility is described, the latter of which was evaluated with isotope-labeled metabolites from a yeast cell extract. The combined experimental and computational advances were found to provide accurate labeling quantitation for metabolic flux analyses.

How Close Are We to Complete Annotation of Metabolomes?

Viant, M.R.; Kurland, I.J.; Jones, M.R.; et al. 

Efforts to identify and annotate an organism's metabolome are continuing through untargeted, one-dimensional and multidimensional, MS-based methodologies. Coordinated advancements have been facilitated by the emergence of international task groups, such as the Model Organism Metabolomes. In this review, Dunn et al. summarize the current status of metabolome characterization, its reporting, and pertinent analytical strategies developed therein.

Plasmonic Silver Nanoshells for Drug and Metabolite Detection

Huang, L.; Wan, J.; Wei, X.; Liu, Y.; et al.

Given the demand for improved analytical methods for biomedical applications, Qian et al. developed plasmonic silver nanoshells for metabolite detection in clinical in vitro diagnostics. The SiO2@Ag nanoshells were first synthesized (by hard templating method) and characterized (through various electron microscopy techniques) to ensure selective analyte trapping prior to high throughput MS screening by laser desorption/ionization. The utility of this designer platform was evaluated in complex biofluids for disease diagnosis (of patients with postoperative brain infection) and therapeutic evaluation (of patients with cerebral edema for pharmacokinetics study), with quantitation enhanced through the use of ¹³C-labeled metabolites.