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1. Evidence of gas-phase pyranose-to-furanose isomerization in protonated peptidoglycans

   https://doi.org/10.1039/D1CP03842G  

   Guan, Shanshan; Bythell, Benjamin J. (October 2021, Physical Chemistry Chemical Physics)

   Peptidoglycans are diverse co- and post-translational modifications of key importance in myriad biological processes. Mass spectrometry is employed to infer their biomolecular sequences and stereochemisties, but little is known about the critical gas-phase dissociation processes involved. Here, using tandem mass spectrometry (MS/MS and MS n ), isotopic labelling and high-level simulations, we identify and characterize a facile isomerization reaction that produces furanose N-acetylated ions. This reaction occurs for both O- and N-linked peptidoglycans irrespective of glycosidic linkage stereochemistry (α/β). Dissociation of the glycosidic and other bonds thus occur from the furanose isomer critically altering the reaction feasibility and product ion structures. 

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2. High-throughput double emulsion-based microfluidic production of hydrogel microspheres with tunable chemical functionalities toward biomolecular conjugation

   https://doi.org/10.1039/c7lc01088e  

   Liu, Eric Y.; Jung, Sukwon; Weitz, David A.; Yi, Hyunmin; Choi, Chang-Hyung (January 2018, Lab on a Chip)

   Chemically functional hydrogel microspheres hold significant potential in a range of applications including biosensing, drug delivery, and tissue engineering due to their high degree of flexibility in imparting a range of functions. In this work, we present a simple, efficient, and high-throughput capillary microfluidic approach for controlled fabrication of monodisperse and chemically functional hydrogel microspheres via formation of double emulsion drops with an ultra-thin oil shell as a sacrificial template. This method utilizes spontaneous dewetting of the oil phase upon polymerization and transfer into aqueous solution, resulting in poly(ethylene glycol) (PEG)-based microspheres containing primary amines (chitosan, CS) or carboxylates (acrylic acid, AA) for chemical functionality. Simple fluorescent labelling of the as-prepared microspheres shows the presence of abundant, uniformly distributed and readily tunable functional groups throughout the microspheres. Furthermore, we show the utility of chitosan's primary amine as an efficient conjugation handle at physiological pH due to its low pKa by direct comparison with other primary amines. We also report the utility of these microspheres in biomolecular conjugation using model fluorescent proteins, R-phycoerythrin (R-PE) and green fluorescent protein (GFPuv), via tetrazine– trans -cyclooctene (Tz–TCO) ligation for CS-PEG microspheres and carbodiimide chemistry for AA-PEG microspheres, respectively. The results show rapid coupling of R-PE with the microspheres' functional groups with minimal non-specific adsorption. In-depth protein conjugation kinetics studies with our microspheres highlight the differences in reaction and diffusion of R-PE with CS-PEG and AA-PEG microspheres. Finally, we demonstrate orthogonal one-pot protein conjugation of R-PE and GFPuv with CS-PEG and AA-PEG microspheres via simple size-based encoding. Combined, these results represent a significant advancement in the rapid and reliable fabrication of monodisperse and chemically functional hydrogel microspheres with tunable properties. 

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3. New Aequorea Fluorescent Proteins for Cell-Free Bioengineering

   https://doi.org/10.1021/acssynbio.3c00057  

   Deich, Christopher; Gaut, Nathaniel J.; Sato, Wakana; Engelhart, Aaron E.; Adamala, Katarzyna P. (April 2023, ACS Synthetic Biology)

   Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing, and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms. 

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4. Rotamer modelling of Cu(II) spin labels based on the double-Histidine motif

   https://doi.org/https://doi.org/10.1007/s00723-018-1052-8  

   Ghosh, Shreya; Saxena, Sunil; Jeschke, Gunnar. (January 2018, Applied magnetic resonance)

   Spin labels attached to two residues of a protein chain have less conformational flexibility than those attached to a single residue and thus lead to a narrower spatialdistribution of the unpaired electron. The case of Cu(II) labels based on the double-histidine (dHis) motif is of particular interest, as it combines the advantage of precise localization of the unpaired electron with a labelling scheme orthogonal to the more common cysteine-based labelling. Here, we introduce an approach for in silico spin labelling of a protein by dHis motifs and Cu(II) complexes of iminodiacetic acid or nitrilotriacetic acid. We discuss a computerized scan for native histidine pairs that might be prone to bind such Cu(II) complexes and spin-labelling site pair scans that can identify suitable double mutants for labelling. Predicted distance distributions between two Cu(II) labels are compared to experimental distance distributions. We also test the hypothesis that elastic network modelling of conformational transitions with Cu2(II)- dHis labels can provide more accurate structural models than with nitroxide labels. 

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5. Metabolomics studies of cell–cell interactions using single cell mass spectrometry combined with fluorescence microscopy

   https://doi.org/10.1039/d2sc02298b  

   Chen, Xingxiu; Peng, Zongkai; Yang, Zhibo (June 2022, Chemical Science)

   Cell–cell interactions are critical for transmitting signals among cells and maintaining their normal functions from the single-cell level to tissues. In cancer studies, interactions between drug-resistant and drug-sensitive cells play an important role in the development of chemotherapy resistance of tumors. As metabolites directly reflect the cell status, metabolomics studies provide insight into cell–cell communication. Mass spectrometry (MS) is a powerful tool for metabolomics studies, and single cell MS (SCMS) analysis can provide unique information for understanding interactions among heterogeneous cells. In the current study, we utilized a direct co-culture system (with cell–cell contact) to study metabolomics of single cells affected by cell–cell interactions in their living status. A fluorescence microscope was utilized to distinguish these two types of cells for SCMS metabolomics studies using the Single-probe SCMS technique under ambient conditions. Our results show that through interactions with drug-resistant cells, drug-sensitive cancer cells acquired significantly increased drug resistance and exhibited drastically altered metabolites. Further investigation found that the increased drug resistance was associated with multiple metabolism regulations in drug-sensitive cells through co-culture such as the upregulation of sphingomyelins lipids and lactic acid and the downregulation of TCA cycle intermediates. The method allows for direct MS metabolomics studies of individual cells labeled with fluorescent proteins or dyes among heterogeneous populations. 

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