Sulfation plays an important role in many metabolic processes such as substrate detoxification, solubilization or activation. The enzymes responsible for these reactions, sulfotransferases, occur in all domains of life and can be classified in two major, evolutionary independent groups, PAPS dependent and PAPS independent or arysulfate sulfotransferases (ASST) enzymes. Besides the fact that they play key roles in biological functions, sulfotransferases can be interesting enzymatic tools to sele…

Discover how biochar enhances nutrient accessibility in compost, driving microbial activity and reducing reliance on chemical fertilizers.

AbstractTop-down proteomics (TDP) enables comprehensive characterization of intact proteoforms, but the analysis of membrane proteoforms remains challenging because of their hydrophobicity and relatively low abundance. To address these challenges, we previously developed Azo, a photocleavable surfactant that enables efficient solubilization and top-down analysis of membrane proteins. Subsequently, we established an Azo-enabled TDP method permitting the comprehensive characterization of phospholamban (PLN), a transmembrane protein that plays crucial roles in calcium handling, directly from cardiac tissue. However, the original workflow required at least 10 mg of human cardiac tissue, making it unsuitable for biopsy-scale and other sample-limited studies. Herein, we developed a streamlined Azo-enabled one-pot extraction strategy for high-sensitivity top-down analysis of PLN that reduces the required tissue input to 1 mg while preserving analytical performance. Using online liquid chromatography–tandem mass spectrometry, we achieved highly reproducible detection, quantification, and post-translational modification localization of endogenous PLN proteoforms from 1 mg of human cardiac tissue. Notably, the streamlined strategy reduced the tissue required for PLN protein extraction 10-fold while retaining the same capacity for comprehensive characterization in the one-pot extraction. Collectively, these results establish a practical strategy for TDP of PLN proteoforms from biopsy-scale and other sample-limited myocardial specimens.

Excipients are widely used to suppress the self-association of therapeutic proteins, yet their mechanisms of action are not well understood and often assumed to be nonspecific. Here we show that ex...

The β2AR construct PN1 was expressed and purified as previously described39,42. In brief, Sf9 cells were infected with a PN1-containing baculovirus produced using the BestBac method. Cells were then collected and resuspended in chilled lysis buffer containing 10 mM HEPES, pH 7.4, 1 mM EDTA, 1 μM alprenolol and protease inhibitors (leupeptin and benzamidine). Lysed cells were then pelleted at 18,600 rpm for 20 min and dounced to homogeneity in chilled solubilization buffer containing 20 mM HEPES pH 7.4, 350 mM NaCl, 1% n-dodecyl β-D-maltoside (DDM), 0.1% cholesteryl hemisuccinate (CHS), 2 mM MgCl2, 1 μM alprenolol, protease inhibitors and benzonase. After stirring for 90 min at 4 °C and centrifugation at 18,600 rpm for 30 min, 2 mM CaCl2 was added to the soluble fraction, which was then applied to anti-Flag (DYKDDDDK) M1 immunoaffinity resin. The receptor was then washed (20 mM HEPES, pH 7.4, 350 mM NaCl, 0.1% DDM, 0.01% CHS, 2 mM CaCl2, leupeptin and benzamidine), eluted (20 mM HEPES, pH 7.4, 350 mM NaCl, 0.1% DDM, 0.01% CHS, 5 mM EDTA and 200 μg ml−1 Flag peptide), and further purified on Superdex 200 10/300 Increase gel filtration column equilibrated in NH buffer (20 mM, HEPES pH 7.4,...

In the context of the global photovoltaic (PV) industry's current period of significant growth and development, the issue of the recycling of waste PV…

Published in World journal of microbiology & biotechnology

Extracellular vesicles (EVs) play a vital role in numerous biological processes. Proteomic research of EVs is crucial for understanding their functions and potential therapeutic implications. Despite ...

Nanoscale hydrophilic-interaction chromatography coupled with tandem mass spectrometry (nanoHILIC/MS/MS) is a promising alternative to reversed-phase liquid chromatography for proteomics, but its appl...

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