Kinome-wide decoding of network-attacking mutations rewiring cancer signaling.

Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks.

Evidence for an RNAi-independent role of Arabidopsis DICER-LIKE2 in growth inhibition and basal antiviral resistance.

Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs, which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense, and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4 since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA.

Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments.

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .

CLIPPER 2.0: Peptide-Level Annotation and Data Analysis for Positional Proteomics.

Positional proteomics methodologies have transformed protease research, and have brought mass spectrometry (MS)-based degradomics studies to the forefront of protease characterization and system-wide interrogation of protease signaling. Considerable advancements in both sensitivity and throughput of liquid chromatography (LC)-MS/MS instrumentation enable the generation of enormous positional proteomics datasets of natural and protein termini and neo-termini of cleaved protease substrates. However, concomitant progress has not been observed to the same extent in data analysis and post-processing steps, arguably constituting the largest bottleneck in positional proteomics workflows. Here, we present a computational tool, CLIPPER 2.0, that builds on prior algorithms developed for MS-based protein termini analysis, facilitating peptide-level annotation and data analysis. CLIPPER 2.0 can be used with several sample preparation workflows and proteomics search algorithms and enables fast and automated database information retrieval, statistical and network analysis, as well as visualization of terminomic datasets. We demonstrate the applicability of our tool by analyzing GluC and MMP9 cleavages in HeLa lysates. CLIPPER 2.0 is available at https://github.com/UadKLab/CLIPPER-2.0.

BloodSpot 3.0: a database of gene and protein expression data in normal and malignant haematopoiesis.

BloodSpot is a specialised database integrating gene expression data from acute myeloid leukaemia (AML) patients related to blood cell development and maturation. The database and interface has helped numerous researchers and clinicians to quickly get an overview of gene expression patterns in healthy and malignant haematopoiesis. Here, we present an update to our framework that includes protein expression data of sorted single cells. With this update we also introduce datasets broadly spanning age groups, which many users have requested, with particular interest for researchers studying paediatric leukaemias. The backend of the database has been rewritten and migrated to a cloud-based environment to accommodate the growth, and provide a better user-experience for our many international users. Users can now enjoy faster transfer speeds and a more responsive interface. In conclusion, the continuing popularity of the database and emergence of new data modalities has prompted us to rewrite and futureproof the back-end, including paediatric centric views, as well as single cell protein data, allowing us to keep the database updated and relevant for the years to come. The database is freely available at www.bloodspot.eu.

A proteomics-based survey reveals thrombospondin-4 as a ligand regulated by the mannose receptor in the injured lung.

Receptor-mediated cellular uptake of specific ligands constitutes an important step in the dynamic regulation of individual protein levels in extracellular fluids. With a focus on the inflammatory lung, we here performed a proteomics-based search for novel ligands regulated by the mannose receptor (MR), a macrophage-expressed endocytic receptor. WT and MR-deficient mice were exposed to lipopolysaccharide, after which the protein content in their lung epithelial lining fluid was compared by tandem mass tag-based mass spectrometry. More than 1200 proteins were identified in the epithelial lining fluid using this unbiased approach, but only six showed a statistically different abundance. Among these, an unexpected potential new ligand, thrombospondin-4 (TSP-4), displayed a striking 17-fold increased abundance in the MR-deficient mice. Experiments using exogenous addition of TSP-4 to MR-transfected CHO cells or MR-positive alveolar macrophages confirmed that TSP-4 is a ligand for MR-dependent endocytosis. Similar studies revealed that the molecular interaction with TSP-4 depends on both the lectin activity and the fibronectin type-II domain of MR and that a closely related member of the TSP family, TSP-5, is also efficiently internalized by the receptor. This was unlike the other members of this protein family, including TSPs -1 and -2, which are ligands for a close MR homologue known as urokinase plasminogen activator receptor-associated protein. Our study shows that MR takes part in the regulation of TSP-4, an important inflammatory component in the injured lung, and that two closely related endocytic receptors, expressed on different cell types, undertake the selective endocytosis of distinct members of the TSP family.

Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics.

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times and high-resolution spectra to quantify the single-cell signal; however, the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time-intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS enhanced quant of single cell spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at an MS2 injection time of 750 ms using a 2 h gradient.

Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application.

Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large-scale biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the most widely applied protease in proteomics due to its characteristics. With ever-larger cohort sizes and possible future clinical application of mass spectrometry-based proteomics, the technical impact of trypsin becomes increasingly relevant. To assess possible biases introduced by trypsin digestion, we evaluated the impact of eight commercially available trypsins in a variety of bottom-up proteomics experiments and across a range of protease concentrations and storage times. To investigate the universal impact of these technical attributes, we included bulk HeLa cell lysate, human plasma, and single HEK293 cells, which were analyzed over a range of selected reaction monitoring (SRM), data-independent acquisition (DIA), and data-dependent acquisition (DDA) instrument methods on three LC-MS instruments. The quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive data set, we report variations between commercial trypsins, their source, and their concentration. Furthermore, we provide suggestions on the handling of trypsin in large-scale studies.

Optimizing Linear Ion-Trap Data-Independent Acquisition toward Single-Cell Proteomics.

A linear ion trap (LIT) is an affordable, robust mass spectrometer that provides fast scanning speed and high sensitivity, where its primary disadvantage is inferior mass accuracy compared to more commonly used time-of-flight or orbitrap (OT) mass analyzers. Previous efforts to utilize the LIT for low-input proteomics analysis still rely on either built-in OTs for collecting precursor data or OT-based library generation. Here, we demonstrate the potential versatility of the LIT for low-input proteomics as a stand-alone mass analyzer for all mass spectrometry (MS) measurements, including library generation. To test this approach, we first optimized LIT data acquisition methods and performed library-free searches with and without entrapment peptides to evaluate both the detection and quantification accuracy. We then generated matrix-matched calibration curves to estimate the lower limit of quantification using only 10 ng of starting material. While LIT-MS1 measurements provided poor quantitative accuracy, LIT-MS2 measurements were quantitatively accurate down to 0.5 ng on the column. Finally, we optimized a suitable strategy for spectral library generation from low-input material, which we used to analyze single-cell samples by LIT-DIA using LIT-based libraries generated from as few as 40 cells.

High Sensitivity Limited Material Proteomics Empowered by Data-Independent Acquisition on Linear Ion Traps.

In recent years, the concept of cell heterogeneity in biology has gained increasing attention, concomitant with a push toward technologies capable of resolving such biological complexity at the molecular level. For single-cell proteomics using Mass Spectrometry (scMS) and low-input proteomics experiments, the sensitivity of an orbitrap mass analyzer can sometimes be limiting. Therefore, low-input proteomics and scMS could benefit from linear ion traps, which provide faster scanning speeds and higher sensitivity than an orbitrap mass analyzer, however at the cost of resolution. We optimized an acquisition method that combines the orbitrap and linear ion trap, as implemented on a tribrid instrument, while taking advantage of the high-field asymmetric waveform ion mobility spectrometry (FAIMS) pro interface, with a prime focus on low-input applications. First, we compared the performance of orbitrap- versus linear ion trap mass analyzers. Subsequently, we optimized critical method parameters for low-input measurement by data-independent acquisition on the linear ion trap mass analyzer. We conclude that linear ion traps mass analyzers combined with FAIMS and Whisper flow chromatography are well-tailored for low-input proteomics experiments, and can simultaneously increase the throughput and sensitivity of large-scale proteomics experiments where limited material is available, such as clinical samples and cellular subpopulations.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing.

Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1ß, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort.

Exploratory Investigation of the Plasma Proteome Associated with the Endotheliopathy of Trauma.

BACKGROUND: The endotheliopathy of trauma (EoT) is associated with increased mortality following injury. Herein, we describe the plasma proteome related to EoT in order to provide insight into the role of the endothelium within the systemic response to trauma. METHODS: 99 subjects requiring the highest level of trauma activation were included in the study. Enzyme-linked immunosorbent assays of endothelial and catecholamine biomarkers were performed on admission plasma samples, as well as untargeted proteome quantification utilizing high-performance liquid chromatography and tandem mass spectrometry. RESULTS: Plasma endothelial and catecholamine biomarker abundance was elevated in EoT. Patients with EoT (n = 62) had an increased incidence of death within 24 h at 21% compared to 3% for non-EoT (n = 37). Proteomic analysis revealed that 52 out of 290 proteins were differentially expressed between the EoT and non-EoT groups. These proteins are involved in endothelial activation, coagulation, inflammation, and oxidative stress, and include known damage-associated molecular patterns (DAMPs) and intracellular proteins specific to several organs. CONCLUSIONS: We report a proteomic profile of EoT suggestive of a surge of DAMPs and inflammation driving nonspecific activation of the endothelial, coagulation, and complement systems with subsequent end-organ damage and poor clinical outcome. These findings support the utility of EoT as an index of cellular injury and delineate protein candidates for therapeutic intervention.

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase.

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059.

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

Comparison of Tenocyte Populations from the Core and Periphery of Equine Tendons.

Tendon is a highly organized, dense connective tissue that has been demonstrated to have very little turnover. In spite of the low turnover, tendon can grow in response to loading, which may take place primarily at the periphery. Tendon injuries and recurrence of injuries are common in both humans and animals in sports. It is unclear why some areas of the tendon are more susceptible to such injuries and whether this is due to intrinsic regional differences in extracellular matrix (ECM) production or tissue turnover. This study aimed to compare populations of tenocytes derived from the tendon core and periphery. Tenocytes were isolated from equine superficial digital flexor tendons (SDFTs), and the proliferation capacity was determined. ECM production was characterized by immuno- and histological staining and by liquid chromatography-mass spectrometry-based proteomics. Core and periphery SDFT cultures exhibited comparable proliferation rates and had very similar proteome profiles, but showed biological variation in collagen type I deposition. In conclusion, the intrinsic properties of tenocytes from different regions of the tendon are very similar, and other factors in the tissue may contribute to how specific areas respond to loading or injury.

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Two strategies were used: the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH-related proteome has been observed with an upregulation of the regulatory subunit of glutamate-cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.

Proteomics identifies differences in fibrotic potential of extracellular vesicles from human tendon and muscle fibroblasts.

BACKGROUND: Fibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis. METHODS: Here, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs. RESULTS: Mass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFß1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix. CONCLUSIONS: Our data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans. Video abstract.