Astrocytes are ubiquitous within the central nervous system (CNS). These cells possess many individual processes which extend out into the neuropil, where they interact with a variety of other cell types, including neurons at synapses. Astrocytes are now known to be active players in all aspects of the synaptic life cycle, including synapse formation and elimination, synapse maturation, maintenance of synaptic homeostasis and modulation of synaptic transmission. Traditionally, astrocytes have been studied as a homogeneous group of cells. However, recent studies have uncovered a surprising degree of heterogeneity in their development and function, suggesting that astrocytes may be matched to neurons to support local circuits. Hence, a better understanding of astrocyte heterogeneity and its implications are needed to understand brain function.

This review aims to summarise the current capabilities of surface mass spectrometry (MS) approaches that offer intact protein analysis, and that of non-covalent complexes. Protein analysis is largely achieved via matrix-assisted laser desorption/ionisation (MALDI), which is in itself a surface analysis approach or solvent-based electrospray ionisation (ESI). Several surface sampling approaches have been developed based on ESI, and those that have been used for intact protein analysis will be discussed below. The extent of protein coverage, top-down elucidation, and probing of protein structure for native proteins and non-covalent complexes will be discussed for each approach. Strategies for improving protein analysis, ranging from sample preparation, and sampling methods to instrument modifications and the inclusion of ion mobility separation in the workflow will also be discussed. The relative benefits and drawbacks of each approach will be summarised, providing an overview of current capabilities.

The plant cell wall is rich in carbohydrates and many fungi and bacteria have evolved to take advantage of this carbon source. These carbohydrates are largely locked away in polysaccharides and so these organisms deploy a range of enzymes that can liberate individual sugars from these challenging substrates. Glycoside hydrolases (GHs) are the enzymes that are largely responsible for bringing about this sugar release; however, 12 years ago, a family of enzymes known as lytic polysaccharide monooxygenases (LPMOs) were also shown to be of key importance in this process. LPMOs are copper-dependent oxidative enzymes that can introduce chain breaks within polysaccharide chains. Initial work demonstrated that they could activate O2 to attack the substrate through a reaction that most likely required multiple electrons to be delivered to the enzyme. More recently, it has emerged that LPMO kinetics are significantly improved if H2O2 is supplied to the enzyme as a cosubstrate instead of O2. Only a single electron is required to activate an LPMO and H2O2 cosubstrate and the enzyme has been shown to catalyse multiple turnovers following the initial one-electron reduction of the copper, which is not possible if O2 is used. This has led to further studies of the roles of the electron donor in LPMO biochemistry, and this review aims to highlight recent findings in this area and consider how ongoing research could impact our understanding of the interplay between redox processes in nature.

Cross-linking mass spectrometry has become an established technology to provide structural information on the topology and dynamics of protein complexes. Readily accessible workflows can provide detailed data on simplified systems, such as purified complexes. However, using this technology to study the structure of protein complexes in situ, such as in organelles, cells, and even tissues, is still a technological frontier. The complexity of these systems remains a considerable challenge, but there have been dramatic improvements in sample handling, data acquisition, and data processing. Here, we summarise these developments and describe the paths towards comprehensive and comparative structural interactomes by cross-linking mass spectrometry.

The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has profoundly changed our current understanding of enzymatic processes underlying the conversion of polysaccharides in the biosphere. LPMOs are truly unique enzymes, harboring a single copper atom in a solvent-exposed active site, allowing them to oxidize C-H bonds at the C1 and/or C4 carbon of glycosidic linkages found in recalcitrant, often crystalline polysaccharides such as cellulose and chitin. To catalyze this challenging reaction, LPMOs harness and control a powerful oxidative reaction that involves Fenton-like chemistry. In this essay, we first draw a brief portrait of the LPMO field, notably explaining the shift from the monooxygenase paradigm (i.e., using O2 as cosubstrate) to that of a peroxygenase (i.e., using H2O2). Then, we briefly review current understanding of how LPMOs generate and control a hydroxyl radical (HO•) generated through Cu(I)-catalyzed H2O2 homolysis, and how this radical is used to create the proposed Cu(II)-oxyl species, abstracting hydrogen atom of the C-H bond. We also point at the complexity of analyzing redox reactions involving reactive oxygen species and address potential deficiencies in the interpretation of existing LPMO data. Being the first copper enzymes shown to enable site-specific Fenton-like chemistry, and maybe not the only ones, LPMOs may serve as a blueprint for future research on monocopper peroxygenases.