An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 µg dry TMT per channel was used to label 6-12 µg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

Investigations into the binding of jadomycin DS to human topoisomerase IIß by WaterLOGSY NMR spectroscopy.

The jadomycins are a family of secondary metabolites produced by S. venezuelae ISP5230. Specific jadomycins have been shown to possess a variety of anticancer, antifungal, and antibacterial properties, with different molecular mechanisms of action. Herein we demonstrate qualitative and quantitative direct binding between the validated anticancer target human topoisomerase IIß and jadomycin DS using WaterLOGSY NMR spectroscopy. Additionally, we report for the first time, that jadomycin DS also binds a variety of other proteins, likely in a non-specific manner. Such interactions may rationalize the potential polypharmacology of jadomycin DS.

Use of Ice Recrystallization Inhibition Assays to Screen for Compounds That Inhibit Ice Recrystallization.

Ice recrystallization inhibition assays are used to screen for compounds that possess the ability to inhibit ice recrystallization. The most common of these assays are the splat cooling assay (SCA) and sucrose sandwich assay (SSA). These two assays possess similarities; however, they vary in their sample size, cooling rate, and the solution used to dissolve the analyte. In this chapter, both assay methods are described in detail, and we perform a direct comparison of the assays by evaluating the IRI activity of an antifreeze protein (AFP I). IRI activity is quantified by using ImageJ software to analyze ice crystals, and a quantitative value describing the efficiency of the inhibitor is generated. This analysis emphasizes the importance of choosing the right assay to measure IRI activity.

Observing enzyme ternary transition state analogue complexes by 19F NMR spectroscopy.

Ternary transition state analogue (TSA) complexes probing the isomerization of ß-d-glucose 1-phosphate (G1P) into d-glucose 6-phosphate (G6P) catalyzed by catalytically active, fluorinated (5-fluorotryptophan), ß-phosphoglucomutase (ßPGM) have been observed directly by 19F NMR spectroscopy. In these complexes MgF3- and AlF4- are surrogates for the transferring phosphate. However, the relevance of these metal fluorides as TSA complexes has been queried. The 1D 19F spectrum of a ternary TSA complex presented a molar equivalence between fluorinated enzyme, metal fluoride and non-isomerizable fluoromethylenephosphonate substrate analogue. Ring flips of the 5-fluoroindole ring remote from the active site were observed by both 19F NMR and X-ray crystallography, but did not perturb function. This data unequivocally demonstrates that the concentration of the metal fluoride complexes is equivalent to the concentration of enzyme and ligand in the TSA complex in aqueous solution.