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Protein footprinting is a useful method for studying protein higher order structure and conformational changes induced by interactions with various ligands via addition of covalent modifications onto the protein. Compared to other methods that provide single amino acid-level structural resolution, such as cryo-EM, X-ray diffraction, and NMR, mass spectrometry (MS)-based methods can be advantageous as they require lower protein amounts and purity. As with other MS-based proteomic methods, such as post-translational modification analysis, enrichment techniques have proven necessary for both optimal sensitivity and sequence coverage when analyzing highly complex proteomes. Currently used reagents for footprinting via covalent labeling, such as hydroxyl radicals and carbodiimide-based methods, do not yet have a suitable enrichment method, limiting their applicability to whole proteome analysis. Here, we report a method for enrichable covalent labeling built upon the GEE/EDC system commonly used to covalently label aspartic acid and glutamic acid residues. Novel labeling reagents containing alkynyl functionality can be "clicked" to any azido-containing molecule with copper-catalyzed azide-alkyne cycloaddition (CuAAC), allowing for enrichment or further labeling. Multiple azide- and alkyne-containing GEE-like molecules were tested, and the most efficient method was determined to be the EDC-facilitated coupling of glycine propargyl amide (GPA) to proteins. The pipeline we report includes clicking via CuAAC to a commercially available biotin-azide containing a photocleavable linker, followed by enrichment using a streptavidin resin and subsequent cleavage under ultraviolet light. The enrichment process was optimized through the screening of clickable amines, coupling reagents, and enrichment scaffolds and methods with pure model proteins and has also been applied to complex mixtures of proteins isolated from the model plant, Arabidopsis thaliana , suggesting that our method may ultimately be used to measure protein conformation on a proteomic scale.

The plasma membrane proton pump is the primary energy transducing, electrogenic ion pump of the plasma membrane in plants and fungi. Compared to its fungal counterpart, the plant plasma membrane proton pump's regulatory C-terminal domain (CTD) contains an additional regulatory segment that links multiple sensory pathways regulating plant cell length through phosphorylation and recruitment of regulatory 14-3-3 proteins. However, a complete structural model of a plant proton pump is lacking. Here, we performed covalent labeling with mass spectrometric analysis (CL-MS) on the Arabidopsis pump AHA2 to identify potential interactions between the CTD and the catalytic domains. Our results suggest that autoinhibition in the plant enzyme is much more structurally complex than in the fungal enzyme., (© 2024 Federation of European Biochemical Societies.)

Premise: A multi-omic approach was used to explore proteins and networks hypothetically important for establishing filament dimorphisms in heterostylous Turnera subulata (Sm.) as an exploratory method to identify genes for future empirical research., Methods: Mass spectrometry (MS) was used to identify differentially expressed proteins and differentially phosphorylated peptides in the developing filaments between the L- and S-morphs. RNAseq was used to generate a co-expression network of the developing filaments, MS data were mapped to the co-expression network to identify hypothetical relationships between the S-gene responsible for filament dimorphisms and differentially expressed proteins., Results: Mapping all MS identified proteins to a co-expression network of the S-morph's developing filaments identified several clusters containing SPH1 and other differentially expressed or phosphorylated proteins. Co-expression analysis clustered CDKG2, a protein that induces endoreduplication, and SPH1-suggesting a shared biological function. MS analysis suggests that the protein is present and phosphorylated only in the S-morph, and thus active only in the S-morph. A series of CDKG2 regulators, including ATM1, and cell cycle regulators also correlated with the presence of reciprocal herkogamy, supporting our interest in the protein., Conclusions: This work has built a foundation for future empirical work, specifically supporting the role of CDKG2 and ATM1 in promoting filament elongation in response to SPH1 perception., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway., (© 2024. The Author(s).)

Hydrophobic microdomains, also known as hydrophobic patches, are essential for many important biological functions of water-soluble proteins. These include ligand or substrate binding, protein-protein interactions, proper folding after translation, and aggregation during denaturation. Unlike transmembrane domains, which are easily recognized from stretches of contiguous hydrophobic sidechains in amino acids via primary protein sequence, these three-dimensional hydrophobic patches cannot be easily predicted. The lack of experimental strategies for directly determining their locations hinders further understanding of their structure and function. Here, we posit that the small triatomic anion N 3 - (azide) is attracted to these patches and, in the presence of an oxidant, forms a radical that covalently modifies C-H bonds of nearby amino acids. Using two model proteins (BSA and lysozyme) and a cell-free lysate from the model higher plant Arabidopsis thaliana , we find that radical-mediated covalent azidylation occurs within buried catalytic active sites and ligand binding sites and exhibits similar behavior to established hydrophobic probes. The results herein suggest a model in which the azido radical is acting as an "affinity reagent" for nonaqueous three-dimensional protein microenvironments and is consistent with both the nonlocalized electron density of the azide moiety and the known high reactivity of azido radicals widely used in organic chemistry syntheses. We propose that the azido radical is a facile means of identifying hydrophobic microenvironments in soluble proteins and, in addition, provides a simple new method for attaching chemical handles to proteins without the need for genetic manipulation or specialized reagents.

We have developed a method for complete dissolution of whole eggs in formic acid that provides a new approach to analyzing egg biomolecules. As expected from prior work with extracted lipids, phosphatidylcholine represents the most abundant 31 P NMR signal. A simplified methanol/chloroform partitioning method for separating the dissolved egg solution into metabolites, lipids and protein was performed and after ultra-high mass resolution and tandem MS fragmentation analyses several phosphatidylcholine molecules containing different fatty acid chain lengths as well as number and position of double bonds was detected. The MS based proteomic analysis further revealed 6 Gallus sequences annotated as 'uncharacterized' because they show no sequence homology with any other protein found in nature and thus, may represent proteins uniquely evolved to perform functions specific to chickens. Overall, this procedure is a rapid and facile means of characterizing in a high throughput and comprehensive manner, the molecular components of whole eggs., Competing Interests: Declaration of Competing Interest The Wisconsin Alumni Research Foundation, which is responsible for the intellectual property arising from UW-Madison research has applied for a patent entitled “Complete dissolution of dried whole egg in formic acid and/or nitric acid and use of the resulting solution in the food industry and for research in human nutrition”., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions-which are more difficult to study-dynamically regulate the proteome's 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based "structural" proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)