Mass Spectrometry Compatible Surfactant for Optimized In-Gel Protein Digestion

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)­undecyloxy)­carbonylamino)­propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5–2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20–30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment

Novel Heterocyclic Analogues of Firefly Luciferin

Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant <i>Photinus pyralis</i> wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479–528 and 518–574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2–9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications

NanoBRETA Novel BRET Platform for the Analysis of Protein–Protein Interactions

Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells