High-Throughput Nanoflow Proteomics Using a Dual-Column Electrospray Source.

With current trends in proteomics, especially regarding clinical and low input (to single cell) samples, it is increasingly important to both maximize the throughput of the analysis and maintain as much sensitivity as possible. The new generation of mass spectrometers (MS) are taking a huge leap in sensitivity, allowing analysis of samples with shorter liquid chromatography (LC) methods while digging as deep in the proteome. However, the throughput can be doubled by implementing a dual column nano-LC-MS configuration. For this purpose, we used a dual-column setup with a two-outlet electrospray source and compared it to a classic dual-column setup with a single-outlet source.

A preclinical platform for assessing long-term drug efficacy exploiting mechanically tunable scaffolds colonized by a three-dimensional tumor microenvironment.

BACKGROUND: Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. METHODS: In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. RESULTS: The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. CONCLUSIONS: The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery.

Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience.

Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Smith (Sm) antigen protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of the mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, as for sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and in enhanced resilience to oxidative stress.

Reduced Levels of Misfolded and Aggregated Mutant p53 by Proteostatic Activation.

In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.

Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease.

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name 'polycation poisoning'. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.

Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling.

SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.

Mass spectrometry and the cellular surfaceome.

The collection of exposed plasma membrane proteins, collectively termed the surfaceome, is involved in multiple vital cellular processes, such as the communication of cells with their surroundings and the regulation of transport across the lipid bilayer. The surfaceome also plays key roles in the immune system by recognizing and presenting antigens, with its possible malfunctioning linked to disease. Surface proteins have long been explored as potential cell markers, disease biomarkers, and therapeutic drug targets. Despite its importance, a detailed study of the surfaceome continues to pose major challenges for mass spectrometry-driven proteomics due to the inherent biophysical characteristics of surface proteins. Their inefficient extraction from hydrophobic membranes to an aqueous medium and their lower abundance compared to intracellular proteins hamper the analysis of surface proteins, which are therefore usually underrepresented in proteomic datasets. To tackle such problems, several innovative analytical methodologies have been developed. This review aims at providing an extensive overview of the different methods for surfaceome analysis, with respective considerations for downstream mass spectrometry-based proteomics.

Binding of Orysata lectin induces an immune response in insect cells.

In mammals, plant lectinshave been shown to possess immunomodulatory properties, acting in both the innate and adaptive immune system to modulate the production of mediators of the immune response, ultimately improving host defences. At present, knowledge of immunomodulatory effects of plant lectins in insects is scarce. Treatment of insect cells with the Orysa sativa lectin, Orysata, was previously reported to induce cell aggregation, mimicking the immune process of encapsulation. In this project we investigated the potential immunomodulatory effects of this mannose-binding lectin using Drosophila melanogaster S2 cells. Identification of the Orysata binding partners on the surface of S2 cells through a pull-down assay and proteomic analysis revealed 221 putative interactors, several of which were immunity-related proteins. Subsequent qPCR analysis revealed the upregulation of Toll- and immune deficiency (IMD)-regulated antimicrobial peptides (Drs, Mtk, AttA, and Dpt) and signal transducers (Rel and Hid) belonging to the IMD pathway. In addition, the iron-binding protein Transferrin 3 was identified as a putative interactor for Orysata, and treatment of S2 cells with Orysata was shown to reduce the intracellular iron concentration. All together, we believe these results offer a new perspective on the effects by which plant lectins influence insect cells and contribute to the study of their immunomodulatory properties.

Macrophage Resistance to Ionizing Radiation Exposure Is Accompanied by Decreased Cathepsin D and Increased Transferrin Receptor 1 Expression.

PURPOSE: To identify a molecular signature of macrophages exposed to clinically relevant ionizing radiation (IR) doses, mirroring radiotherapy sessions. METHODS: Human monocyte-derived macrophages were exposed to 2 Gy/ fraction/ day for 5 days, mimicking one week of cancer patient's radiotherapy. Protein expression profile by proteomics was performed. RESULTS: A gene ontology analysis revealed that radiation-induced protein changes are associated with metabolic alterations, which were further supported by a reduction of both cellular ATP levels and glucose uptake. Most of the radiation-induced deregulated targets exhibited a decreased expression, as was the case of cathepsin D, a lysosomal protease associated with cell death, which was validated by Western blot. We also found that irradiated macrophages exhibited an increased expression of the transferrin receptor 1 (TfR1), which is responsible for the uptake of transferrin-bound iron. TfR1 upregulation was also found in tumor-associated mouse macrophages upon tumor irradiation. In vitro irradiated macrophages also presented a trend for increased divalent metal transporter 1 (DMT1), which transports iron from the endosome to the cytosol, and a significant increase in iron release. CONCLUSIONS: Irradiated macrophages present lower ATP levels and glucose uptake, and exhibit decreased cathepsin D expression, while increasing TfR1 expression and altering iron metabolism.

The lectin Orysata induces phosphatase-mediated and carbohydrate-independent aggregation of insect cells.

Lectins, or carbohydrate-binding proteins, can cause agglutination of particular cells. This process is mediated by the interaction of the carbohydrate-binding domain with sugar structures on the cell surface, and this binding can be inhibited by pre-incubation of the lectin with its specific sugars. However, when incubated with insect cells, Orysata, a mannose-binding lectin from rice, caused aggregation of the cells, independent from carbohydrate binding activity. This phenomenon was observed for multiple insect cell lines, confirming the robustness of this phenotype. While the carbohydrate-dependent agglutination of red blood cells happens within minutes, the carbohydrate-independent aggregation of insect cells requires longer incubation times. Further analysis with the galactose-binding lectins SSA and Jacalin, validated the robustness of this lectin-induced, carbohydrate-independent aggregation in different insect cell lines. Since proteomic analysis revealed no changes in the proteome after treatment with the lectins, this cell aggregation is likely caused by the (in) activation or re-organization of the existing surface proteins. The use of inhibitors of phosphorylation and dephosphorylation, staurosporine (STS) and a phosphatase inhibitor (PPI) cocktail, pointed to dephosphorylation as a key mechanism in the lectin-induced, carbohydrate-independent aggregation of insect cells. Similar to contact inhibition, cell proliferation in cell aggregates was decreased. Analysis of the marker for cell proliferation, cyclin E, confirmed that aggregated cells enter a quiescent state. The current data offer a new perspective on the mechanism by which lectins execute their activities, specifically through lectin-induced phosphatase-mediated cell aggregation and proliferation inhibition, independent from their carbohydrate-binding activity.

Simple Peptide Quantification Approach for MS-Based Proteomics Quality Control.

Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with the sample preparation protocol, in particular, after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in the MS-loading solvent, consuming only 2 µL of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 µg as an optimal peptide amount to be injected into this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.

N-glycosylation Site Analysis Reveals Sex-related Differences in Protein N-glycosylation in the Rice Brown Planthopper (Nilaparvata lugens).

Glycosylation is a common modification of proteins and critical for a wide range of biological processes. Differences in protein glycosylation between sexes have already been observed in humans, nematodes and trematodes, and have recently also been reported in the rice pest insect Nilaparvata lugens Although protein N-glycosylation in insects is nowadays of high interest because of its potential for exploitation in pest control strategies, the functionality of differential N-glycosylation between sexes is yet unknown. In this study, therefore, the occurrence and role of sex-related protein N-glycosylation in insects were examined. A comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes at the glycopeptide level. N-glycopeptide enrichment via lectin capturing using the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans, resulted in the identification of over 1300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosylation sites were shown to be derived from proteins with a putative role in insect reproduction. In addition, differential glycan composition between males and females was observed for proteins shared across sexes. Both lectin blotting experiments as well as transcript expression analyses with complete insects and insect tissues confirmed the observed differences in N-glycosylation of proteins between sexes. In conclusion, this study provides further evidence for protein N-glycosylation to be sex-related in insects. Furthermore, original data on N-glycosylation sites of N. lugens adults are presented, providing novel insights into planthopper's biology and information for future biological pest control strategies.

Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress.

BACKGROUND: Fully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of H2O2 in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14. METHODS: The proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14. With cross-linking, microscale thermophoresis, and active-site titration kinetics, the interaction and influence of ERD14 on the function of two target proteins: glutathione transferase Phi9 and catalase was examined. RESULTS: Under osmotic stress, redox enzymes, specifically the glutathione transferase Phi enzymes, are upregulated. Using microscale thermophoresis, we showed that ERD14 directly interacts with GSTF9 with a KD of ~25 µM. ERD14 activates the inactive GSTF9 molecules, protects GSTF9 from oxidation, and can also increases the activity of the enzyme. Aside from GSTF9, we found that ERD14 can also interact with catalase, an important cellular H2O2 scavenging enzyme, with a KD of ~0.13 µM, and protects it from dehydration-induced loss of activity. CONCLUSIONS: We propose that fully intrinsically disordered dehydrin ERD14 might protect and even activate redox enzymes, helping plants to survive oxidative stress under dehydration conditions. GENERAL SIGNIFICANCE: ERD14 has a direct effect on the activity of redox enzymes.

Distinct branches of the N-end rule pathway modulate the plant immune response.

The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.

A Well-Controlled BioID Design for Endogenous Bait Proteins.

The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate the overexpression of bait proteins. Control cell lines are typically nonengineered cells or engineered clones, implying a considerable risk for artifacts because of clonal variation. Genome engineering can also transform BioID, a proximity labeling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, resulting in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins wherein we introduce a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering, leading to bicistronic expression of both p53 and BirA* under control of the endogenous promoter. By targeting a Cas9-cytidine deaminase base editor to the T2A autocleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. Using quantitative proteomics we show significant benefits over the classical ectopic expression of p53-BirA*, and we provide a first well-controlled view of the proximal proteins of endogenous p53 in colon carcinoma cells. This novel application for base editors expands the CRISPR/Cas9 toolbox and can be a valuable addition for synthetic biology.

Challenges in the Structural-Functional Characterization of Multidomain, Partially Disordered Proteins CBP and p300: Preparing Native Proteins and Developing Nanobody Tools.

The structural and functional characterization of large multidomain signaling proteins containing long disordered linker regions represents special methodological and conceptual challenges. These proteins show extreme structural heterogeneity and have complex posttranslational modification patterns, due to which traditional structural biology techniques provide results that are often difficult to interpret. As demonstrated through the example of two such multidomain proteins, CREB-binding protein (CBP) and its paralogue, p300, even the expression and purification of such proteins are compromised by their extreme proteolytic sensitivity and structural heterogeneity. In this chapter, we describe the effective expression of CBP and p300 in a eukaryotic host, Sf9 insect cells, followed by their tandem affinity purification based on two terminal tags to ensure their structural integrity. The major focus of this chapter is on the development of novel accessory tools, single-domain camelid antibodies (nanobodies), for structural-functional characterization. Specific nanobodies against full-length CBP and p300 can specifically target their different regions and can be used for their marking, labeling, and structural stabilization in a broad range of in vitro and in vivo studies. Here, we describe four high-affinity nanobodies binding to the KIX and the HAT domains, either mimicking known interacting partners or revealing new functionally relevant conformations. As immunization of llamas results in nanobody libraries with a great sequence variation, deep sequencing and interaction analysis with different regions of the proteins provide a novel approach toward developing a panel of specific nanobodies.

A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

DYn-2 Based Identification of Arabidopsis Sulfenomes.

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.