Endogenous Production and Vibrational Analysis of Heavy-Isotope-Labeled Peptides from Cyanobacteria.

Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass-sensitive measurement techniques. In this study, a cyanobacterium was cultured in 15 N/13 C-enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC-MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM-IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin-LR (MCLR), cyanopeptolin-A (CYPA), and aerucyclamide-A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy-labeled natural products. Manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy-labeled nutrients provides an efficient means of producing heavy-labeled natural products for mass-sensitive imaging studies.

Three-in-one method for high throughput plant multi-omics.

Multi-omics has gained momentum over the past few years especially in plant single cell-type analysis as they aim to understand cellular molecular networks across different levels of genetic information flow. For multi-omics sample preparation, molecular extractions performed non-simultaneously create rooms for variation, inaccurate data, waste of limited samples, resources and labor. Here we optimized a protocol for 3-in-1 simultaneous extraction of RNA, metabolites, and proteins from the same single cell-type sample. We adapted a commercially available RNA kit with a few modifications to obtain high quality starting materials for sequencing and LC-MS/MS-based metabolomics and proteomics. RNAs are bound to the column, metabolites were extracted in a polar solvent and proteins are precipitated using acetone. This creates an all-in-one workflow using a standard RNA kit. Little training is required to carry out this protocol as it is simple and easy to use. It may be used with a wide range of plant species and different amounts of starting materials, including single cells.

Creation of a Plant Metabolite Spectral Library for Untargeted and Targeted Metabolomics.

Large-scale high throughput metabolomic technologies are indispensable components of systems biology in terms of discovering and defining the metabolite parts of the system. However, the lack of a plant metabolite spectral library limits the metabolite identification of plant metabolomic studies. Here, we have created a plant metabolite spectral library using 544 authentic standards, which increased the efficiency of identification for untargeted metabolomic studies. The process of creating the spectral library was described, and the mzVault library was deposited in the public repository for free download. Furthermore, based on the spectral library, we describe a process of creating a pseudo-targeted method, which was applied to a proof-of-concept study of Arabidopsis leaf extracts. As authentic standards become available, more metabolite spectra can be easily incorporated into the spectral library to improve the mzVault package.

Proteomics and phosphoproteomics of C3 to CAM transition in the common ice plant.

Among all post-translational modifications of proteins, phosphorylation is one of the most common and most studied. Since plants are sessile organisms, many physiological processes on which their survival depends are regulated by phosphorylation and dephosphorylation. Understanding the extent to which a plant proteome is phosphorylated at specific developmental stages and/or under certain environmental conditions is essential for identifying molecular switches that regulate physiological processes and responses. While most phosphoproteomic workflows proposed in the literature provide tools to exclusively analyze phosphorylated proteins, it is imperative to examine both the proteome and the phosphoproteome to reveal the true complexity of a biological process. Here we describe a mass spectrometry-based phosphoproteomics workflow to analyze both total and phosphorylated proteins. Our method includes phenol-based protein extraction, as well as techniques to measure the quantity and quality of protein extracts. In addition, we compare in detail the efficiency and suitability of in-gel and in-solution trypsin digestion methods. A metal oxide affinity chromatography technique for rapid and efficient enrichment of phosphorylated peptides and an LC-MS/MS method for analysis of the phosphorylated peptides are described. Finally, we present and discuss the results generated by applying this workflow to our study of the C3 to CAM transition in the common ice plant (Mesembryanthemum crystallinum). Overall, our workflow provides robust methods for the identification of phosphoproteins and total proteins. It can be broadly applied to many other organisms and sample types, and the results provide a more accurate picture of the molecular switches that regulate different biological processes.

Identification of MPK4 kinase interactome using TurboID proximity labeling proteomics in Arabidopsis thaliana.

TurboID is a new and efficient proximity labeling system that was first developed in living mammalian cells. TurboID is a modified bacterial biotin ligase that can be fused to a bait protein, which can then modify proximal interacting proteins with biotin. Prey proteins subsequently labeled with biotin tags will be pulled down with streptavidin-coated beads and identified by mass spectrometry-based proteomics. TurboID has been recently applied to living plant cells and provided promising results in identification of interacting proteins. Mitogen-activated protein kinase 4 (MPK4) is important for plant growth, development, and defense; however, the molecular mechanisms underlying the range of MPK4 functions are not completely known. Here we use modern proteomics together with the TurboID in a proof-of-concept study to profile the MPK4 interactome and uncover the functions of MPK4 in plant signaling cascades.

Mitogen-Activated Protein Kinase 4-Regulated Metabolic Networks.

Mitogen-activated protein kinase 4 (MPK4) was first identified as a negative regulator of systemic acquired resistance. It is also an important kinase involved in many other biological processes in plants, including cytokinesis, reproduction, and photosynthesis. Arabidopsis thaliana mpk4 mutant is dwarf and sterile. Previous omics studies including genomics, transcriptomics, and proteomics have revealed new functions of MPK4 in different biological processes. However, due to challenges in metabolomics, no study has touched upon the metabolomic profiles of the mpk4 mutant. What metabolites and metabolic pathways are potentially regulated by MPK4 are not known. Metabolites are crucial components of plants, and they play important roles in plant growth and development, signaling, and defense. Here we used targeted and untargeted metabolomics to profile metabolites in the wild type and the mpk4 mutant. We found that in addition to the jasmonic acid and salicylic acid pathways, MPK4 is involved in polyamine synthesis and photosynthesis. In addition, we also conducted label-free proteomics of the two genotypes. The integration of metabolomics and proteomics data allows for an insight into the metabolomic networks that are potentially regulated by MPK4.

Integrative Proteomic and Phosphoproteomic Analyses of Pattern- and Effector-Triggered Immunity in Tomato.

Plants have evolved a two-layered immune system consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI and ETI are functionally linked, but also have distinct characteristics. Unraveling how these immune systems coordinate plant responses against pathogens is crucial for understanding the regulatory mechanisms underlying plant defense. Here we report integrative proteomic and phosphoproteomic analyses of the tomato-Pseudomonas syringae (Pst) pathosystem with different Pst mutants that allow the dissection of PTI and ETI. A total of 225 proteins and 79 phosphopeptides differentially accumulated in tomato leaves during Pst infection. The abundances of many proteins and phosphoproteins changed during PTI or ETI, and some responses were triggered by both PTI and ETI. For most proteins, the ETI response was more robust than the PTI response. The patterns of protein abundance and phosphorylation changes revealed key regulators involved in Ca2+ signaling, mitogen-activated protein kinase cascades, reversible protein phosphorylation, reactive oxygen species (ROS) and redox homeostasis, transcription and protein turnover, transport and trafficking, cell wall remodeling, hormone biosynthesis and signaling, suggesting their common or specific roles in PTI and/or ETI. A NAC (NAM, ATAF, and CUC family) domain protein and lipid particle serine esterase, two PTI-specific genes identified from previous transcriptomic work, were not detected as differentially regulated at the protein level and were not induced by PTI. Based on integrative transcriptomics and proteomics data, as well as qRT-PCR analysis, several potential PTI and ETI-specific markers are proposed. These results provide insights into the regulatory mechanisms underlying PTI and ETI in the tomato-Pst pathosystem, and will promote future validation and application of the disease biomarkers in plant defense.

Primary angle closure glaucoma is characterized by altered extracellular matrix homeostasis in the iris.

PURPOSE: To characterize the proteome of the iris in primary angle closure glaucoma (PACG). EXPERIMENTAL DESIGN: In this cross-sectional study, iris samples were obtained from surgical iridectomy of 48 adults with PACG and five normal controls. Peptides from iris were analysed using liquid chromatography-tandem mass spectrometry on an Orbitrap Q Exactive Plus mass spectrometer. Verification of proteins of interest was conducted using selected reaction monitoring on a triple quadrupole mass spectrometer. The main outcome was proteins with a log2 two-fold difference in expression in iris between PACG and controls. RESULTS: There were 3,446 non-redundant proteins identified in human iris, of which 416 proteins were upregulated and 251 proteins were downregulated in PACG compared with controls. Thirty-two upregulated proteins were either components of the extracellular matrix (ECM) (fibrillar collagens, EMILIN-2, fibrinogen, fibronectin, matrilin-2), matricellular proteins (thrombospondin-1), proteins involved in cell-matrix interactions (integrins, laminin, histidine-rich glycoprotein, paxillin), or protease inhibitors known to modulate ECM turnover (α-2 macroglobulin, tissue factor pathway inhibitor 2, papilin). Two giant proteins, titin and obscurin, were up- and down-regulated, respectively, in the iris in PACG compared with controls. CONCLUSIONS AND CLINICAL RELEVANCE: This proteomic study shows that ECM composition and homeostasis are altered in the iris in PACG.

Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses.

Adeno-associated viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, post-translational modifications (PTMs), and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized peptide mapping-based workflow that provides thorough characterization within 1 day. This workflow is also highly reproducible due to its simplicity having very few steps and is easy to perform proteolytic digestion utilizing thermally stable pepsin, which is active at 70 °C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low-abundance VP1 capsid protein with a digestion process taking only 10 min to prepare and 45 min to complete the digestion.

Combined ultraviolet and darkness regulation of medicinal metabolites in Mahonia bealei revealed by proteomics and metabolomics.

Roots of Mahonia bealei have been used as traditional Chinese medicine with antibacterial, antioxidant and anti-inflammatory properties due to its high alkaloid content. Previously, we reported that alkaloid and flavonoid contents in the M. bealei leaves could be increased by the combined ultraviolet B and dark treatment (UV+D). To explore the underlying metabolic pathways and networks, proteomic and metabolomic analyses of the M. bealei leaves were conducted. Proteins related to tricarboxylic acid cycle, transport and signaling varied greatly under the UV + D. Among them, calmodulin involved in calcium signaling and ATP-binding cassette transporter involved in transport of berberine were increased. Significantly changed metabolites were overrepresented in phenylalanine metabolism, nitrogen metabolism, phenylpropanoid, flavonoid and alkaloid biosynthesis. In addition, the levels of salicylic acid and gibberellin decreased in the UV group and increased in the UV + D group. These results indicate that multi-hormone crosstalk may regulate the biosynthesis of flavonoids and alkaloids to alleviate oxidative stress caused by the UV + D treatment. Furthermore, protoberberine alkaloids may be induced through calcium signaling crosstalk with reaction oxygen species and transported to leaves. SIGNIFICANCE: Mahonia bealei root and stem, not leaf, were used as traditional medicine for a long history because of the high contents of active components. In the present study, UV-B combined with dark treatments induced the production of alkaloids and flavonoids in the M. bealei leaf, especially protoberberine alkaloids such as berberine. Multi-omics analyses indicated that multi-hormone crosstalk, enhanced tricarboxylic acid cycle and active calcium signaling were involved. The study informs a strategy for utilization of the leaves, and improves understanding of the functions of secondary metabolites in M. bealei.

Identification of DIR1-Dependant Cellular Responses in Guard Cell Systemic Acquired Resistance.

After localized invasion by bacterial pathogens, systemic acquired resistance (SAR) is induced in uninfected plant tissues, resulting in enhanced defense against a broad range of pathogens. Although SAR requires mobilization of signaling molecules via the plant vasculature, the specific molecular mechanisms remain elusive. The lipid transfer protein defective in induced resistance 1 (DIR1) was identified in Arabidopsis thaliana by screening for mutants that were defective in SAR. Here, we demonstrate that stomatal response to pathogens is altered in systemic leaves by SAR, and this guard cell SAR defense requires DIR1. Using a multi-omics approach, we have determined potential SAR signaling mechanisms specific for guard cells in systemic leaves by profiling metabolite, lipid, and protein differences between guard cells in the wild type and dir1-1 mutant during SAR. We identified two long-chain 18 C and 22 C fatty acids and two 16 C wax esters as putative SAR-related molecules dependent on DIR1. Proteins and metabolites related to amino acid biosynthesis and response to stimulus were also changed in guard cells of dir1-1 compared to the wild type. Identification of guard cell-specific SAR-related molecules may lead to new avenues of genetic modification/molecular breeding for disease-resistant plants.

Comparative proteomics of Mesembryanthemum crystallinum guard cells and mesophyll cells in transition from C3 to CAM.

Salinity can induce Mesembryanthemum crystallinum to shift its photosynthesis from C3 to crassulacean acid metabolism (CAM), leading to enhanced plant water use efficiency. Studying how M. crystallinum changes its carbon fixation pathways is important for potential translation into crops and enhancing crop resilience. In this study, we examined proteomic changes in guard cells and mesophyll cells in the course of the C3 to CAM transition. We collected enriched guard cells and mesophyll cells during a short period of transition. A total of 1153 proteins were identified and quantified in the two cell-types. During the transition, proteins in the guard cells and mesophyll cells exhibited differential changes. For example, we observed nocturnal carbon fixation in mesophyll cells and proteins involved in cell growth in the two cell-types. Proteins involved in osmotic adjustment, ion transport, energy metabolism and light response may play important roles in the C3 to CAM transition. Real-time PCR experiments were conducted to determine potential correlations between transcript and protein levels. These results have highlighted potential molecular mechanisms underlying the C3 to CAM transition of guard cells and mesophyll cells of the important facultative CAM plant. BIOLOGICAL SIGNIFICANCE: Fresh water resource for agricultural food production is a global challenge. Nature has evolved crassulacean acid metabolism (CAM) plants with enhanced water use efficiency. Using single cell-type proteomics, this study revealed molecular changes taking place in guard cells and mesophyll cells during the shift of ice plant photosynthesis from C3 to CAM. The results have provided important insights into the CAM transition and may facilitate effort toward enhancing crop resilience for global food security.

Identification and Recent Approaches for Evaluation and Management of Rehabilitation Concerns for Patients with Freeman-Burian Syndrome: Principles for Global Treatment.

Freeman-Burian syndrome, formerly Freeman-Sheldon syndrome, is a rare congenital complex myopathic craniofacial syndrome that frequently involves extremity joint deformities, abnormal spinal curvatures, and chest wall mechanical problems that, together with spinal deformities, impair pulmonary function. As part of a clinical practice guideline development, we evaluated 19 rehabilitation-related articles from our formal systematic review, and from these and our experience, we describe rehabilitation considerations. Research in this area has widespread methodologic problems. While many challenges are present, much can be done to afford these patients a good quality of life through careful planning.