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Sensitivity to molecular ions remains a limiting factor for high resolution imaging mass spectrometry of organic and biological materials. Here, we investigate a variant of matrix-enhanced secondary ion mass spectrometry in which the transfer of matrix molecules to the analyte sample is carried out in situ (in situ ME-SIMS). This approach is therefore compatible with both 2D and 3D imaging by SIMS. In this exploratory study, nanoscale matrix layers were sputter-transferred inside our time-of-flight (ToF)-SIMS to a series of thin films of biomolecules (proteins, sugars, lipids) adsorbed on silicon, and the resulting layers were analyzed and depth-profiled. For this purpose, matrix molecules were desorbed from a coated target (obtained by drop-casting or sublimation) using 10 keV Ar-3000(+) ion beam sputtering, followed by redeposition on a collector carrying the sample to be analyzed. After evaluating the quality of the transfer of six different matrices on bare Si collectors, alpha-cyano-4-hydroxycinnamic acid (CHCA) was selected for further experiments. The mass spectra and depth profiles obtained from the organic layer prior to and after the sputter-transfer of CHCA were compared, along with those obtained from regular ME-SIMS samples (dried droplets) and, finally, with MALDI data for the same matrix-analyte combinations. Signal amplification factors were calculated by dividing the integrated molecular intensities obtained with or without matrix transfer. While the amplification factors are between 0.5 and 2 for molecules already detected with high intensities in SIMS, such as cholesterol or human angiotensin, other compounds show very large integrated signal amplification, even above two orders of magnitude. This is the case for d-glucose and cardiolipin, for which the molecular ion intensity is low (or very low) under normal SIMS analysis conditions. For such low ionization probability compounds, the beneficial effect of the matrix is unquestionable. Test experiments on mouse brain tissue sections also indicate signal enhancement with the matrix, especially for high mass lipid ions.

Introduction Antibody-mediated rejection (ABMR) impacts kidney allograft outcome. The diagnosis is made based on findings from invasive kidney transplant biopsy specimens. The aim of this study was to identify a noninvasive urinary protein biomarker for ABMR after kidney transplantation. Methods We performed a multicenter case-control study to identify a urinary biomarker for ABMR (training cohort, n = 249) and an independent, prospective multicenter cohort study for validation (n = 391). We used concomitant biopsies to classify the samples according to the Banff classification. After untargeted protein identification and quantification, we used a support vector machine to train the model in the training cohort. The primary endpoint was the diagnostic accuracy of the urinary biomarker for ABMR in the validation cohort. Results We identified a set of 10 urinary proteins that accurately discriminated patients with (n = 60) and without (n = 189) ABMR in the training cohort with an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.96–1.00). The diagnostic accuracy was maintained in the validation cohort (AUC, 0.88; 95% CI, 0.8–0.93) for discriminating the presence (n = 43) from the absence (n = 348) of ABMR. The negative predictive value of the 10-protein marker set for exclusion of ABMR was 0.99, and the positive predictive value was 0.33. The diagnostic accuracy was independent of the reason for performing the biopsy, time after transplantation, and better than the accuracy of gross proteinuria (AUC, 0.76). Conclusions We identified and validated a urinary protein biomarker set that can be used to exclude ABMR., Graphical abstract

Introduction: Ventilator-associated pneumonia (VAP) caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in hospital intensive care units (ICU). Rapid identification of P. aeruginosa-derived markers in easily accessible patients’ samples can enable an early detection of P. aeruginosa VAP (VAP-PA), thereby stewarding antibiotic use and improving clinical outcomes. Methods: Metabolites were analysed using liquid chromatography-mass spectrometry (LC-MS) in prospectively collected urine samples from mechanically ventilated patients admitted to the Antwerp University Hospital ICU. Patients were followed from the start of mechanical ventilation (n = 100 patients) till the time of clinical diagnosis of VAP (n = 13). Patients (n = 8) in whom diagnosis of VAP was further confirmed by culturing respiratory samples and urine samples were studied for semi-quantitative metabolomics. Results: We first show that multivariate analyses highly discriminated VAP-PA from VAP–non-PA as well as from the pre-infection groups ( R2 = .97 and .98, respectively). A further univariate analysis identified 58 metabolites that were significantly elevated or uniquely present in VAP-PA compared to the VAP–non-PA and pre-infection groups ( P Conclusions: Considerable differences exist between urine metabolites in VAP-PA compared to VAP due to other bacterial aetiologies as well to non-VAP (pre-infection) timepoints. The unique urinary metabolic biomarkers we describe here, if further validated, could serve as highly specific diagnostic biomarkers of VAP-PA.

Antimicrobial peptides (AMPs) are known best for their role in innate immunity against bacteria, viruses, parasites and fungi. However, not only are they showing increasing promise as potential antimicrobial drug candidates, recently, it has been reported that certain AMPs also show a cytotoxic effect against cancer cells. Their possible antitumor effect could make AMPs interesting candidate cancer biomarkers and a possible lead for new anticancer therapy. Due to their cyclic structure, detection and identification of AMPs is challenging, however, mass spectrometry (imaging; MSI) has been shown as a powerful tool for visualization and identification of (unknown) cyclic AMPs. In this chapter, we will discuss how mass spectrometry (imaging), combined with the use of electron-transfer dissociation (ETD) as fragmentation technique, can be used as a reliable method to identify AMPs in their native cyclic state. Using this approach, we have previously detected and identified human neutrophil peptides (HNPs) as important AMPs in cancer, of which a detailed bacterial, viral and cancer-related overview will be presented.

Ventilator-associated pneumonia (VAP) caused byMetabolites were analysed using liquid chromatography-mass spectrometry (LC-MS) in prospectively collected urine samples from mechanically ventilated patients admitted to the Antwerp University Hospital ICU. Patients were followed from the start of mechanical ventilation (n = 100 patients) till the time of clinical diagnosis of VAP (n = 13). Patients (n = 8) in whom diagnosis of VAP was further confirmed by culturing respiratory samples and urine samples were studied for semi-quantitative metabolomics.We first show that multivariate analyses highly discriminated VAP-PA from VAP-non-PA as well as from the pre-infection groups (Considerable differences exist between urine metabolites in VAP-PA compared to VAP due to other bacterial aetiologies as well to non-VAP (pre-infection) timepoints. The unique urinary metabolic biomarkers we describe here, if further validated, could serve as highly specific diagnostic biomarkers of VAP-PA.

Sensitivity to molecular ions remains a limiting factor for high resolution imaging mass spectrometry of organic and biological materials. Here, we investigate a variant of matrix-enhanced secondary ion mass spectrometry in which the transfer of matrix molecules to the analyte sample is carried out

Aneuploidy causes broad-scale disruption in the stochiometric balances of transcripts, proteins, and metabolites. These disruptions often lead to detrimental effects for the organism, but for some organisms and in certain environments, it can also cause a fitness advantage. Understanding the complex trade-off between aneuploidy fitness gains and losses requires a systems biological comprehension of its molecular impact. The protozoan parasite Leishmania provides a unique study angle to this problem, as the pathogen lacks transcriptional regulation of individual protein-coding genes and has an unusually high tolerance for aneuploidy. Here, we present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. This 4 layer multi-omics analysis unambiguously establishes that despite this tolerance, aneuploidy in Leishmania globally and drastically impacts the transcriptome and proteome of affected chromosomes, ultimately explaining the degree of metabolic differences between strains. This impact appears to be present throughout the parasite’s life cycle as we observed it in both its proliferative and infectious life stages. We show that, through dosage compensation, protein complex subunits, secreted proteins, and non-cytoplasmic proteins responded less or even not at all to aneuploidy. In contrast to other Eukaryotes, we did not observe the widespread regulation at the transcript level that typically modulate some of the negative effects of aneuploidy. Instead, Leishmania features a surprisingly high number of regulated proteins encoded by non-aneuploid chromosomes, suggesting that aneuploidy results in extensive post-transcriptionally modulation of protein levels. Our results provide the foundation for the integrated molecular understanding of aneuploidy in Leishmania. They also highlight that the parasite is an attractive model to study processes of post-transcriptional modulation of protein levels in the context of aneuploidy and beyond.

Rationale: The current methods for identifying peptides in mass spectral product ion data still struggle to do so for the majority of spectra. Based on the experimental setup and other assumptions, such methods restrict the search space to speed up computations, but at the cost of creating blind spots. The proteomics community would greatly benefit from a method that is capable of covering the entire search space without using any restrictions, thus establishing a baseline for identification. Methods: We conceived the "mass pattern paradigm" (MPP) that enables the creation of such an identification method, and we implemented it into a prototype database search engine "PRiSM" (PRotein-Spectrum Matching). We then assessed its operational characteristics by applying it to publicly available high-precision mass spectra of low and high identification difficulty. We used those characteristics to gain theoretical insights into trade-offs between sensitivity and speed when trying to establish a baseline for identification. Results: Of 100 low difficulty spectra, PRiSM and SEQUEST agree on 84 identifications (of which 75 are statistically significant). Of 15 of 100 spectra not identified in a previous study (using SEQUEST), 13 are considered reliable after visual inspection and represent 3 proteins (out of 9 in total) not detected previously. Conclusions: Despite leaving noise intact, the simple PRiSM prototype can make statistically reliable identifications, while controlling the false discovery rate by fitting a null distribution. It also identifies some spectra previously unidentifiable in an "extremely open" SEQUEST search, paving the way to establishing a baseline for identification in proteomics. Department of Economy, Science and Innovation, Flanders; Vlaamse Instelling voor Technologisch Onderzoek, Grant/Award Number: UH_PhD_1701

We analysed the cellular and molecular changes in the leaf growth zone of tolerant and sensitive rice varieties in response to suboptimal temperatures. Cold reduced the final leaf length by 35% and 51% in tolerant and sensitive varieties, respectively. Tolerant lines exhibited a smaller reduction of the leaf elongation rate and greater compensation by an increased duration of leaf growth. Kinematic analysis showed that cold reduced cell production in the meristem and the expansion rate in the elongation zone, but the latter was compensated for by a doubling of the duration of cell expansion. We performed iTRAQ proteome analysis on proliferating and expanding parts of the leaf growth zone. We identified 559 and 542 proteins, of which 163 and 210 were differentially expressed between zones, and 96 and 68 between treatments, in the tolerant and sensitive lines, respectively. The categories protein biosynthesis and redox homeostasis were significantly overrepresented in the up-regulated proteins. We therefore measured redox metabolites and enzyme activities in the leaf growth zone, demonstrating that tolerance of rice lines to suboptimal temperatures correlates with the ability to up-regulate enzymatic antioxidants in the meristem and non-enzymatic antioxidants in the elongation zone. Fil: Gázquez, Ayelén. Universiteit Antwerp; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Abdelgawad, Hamada. Universiteit Antwerp; Bélgica Fil: Baggerman, Geert. Universiteit Antwerp; Bélgica Fil: Van Raemdonck, Geert. Universiteit Antwerp; Bélgica Fil: Asard, Han. Universiteit Antwerp; Bélgica Fil: Maiale, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Rodriguez, Andres Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Beemster, Gerrit T. S.. Universiteit Antwerp; Bélgica

© 2019 Elsevier B.V. This study has focussed on the effect of low temperature storage on the postharvest ripening of green tomatoes; more specifically, on the ethylene signal transduction in relation to the ethylene biosynthesis pathway. To this end, the first elements of the ethylene signal transduction, ethylene receptors, CTR and EIN2 have been analysed in tomatoes stored at three different temperatures in terms of both their gene and protein expression levels. These results have been related to quality changes and enzymes and intermediates of the ethylene biosynthesis pathway to better understand the ethylene control of ripening under low temperatures. The first changes observed during low temperature storage took place at the ethylene biosynthesis level, which might enhance changes in the ethylene signal transduction. Although expression of most ethylene signalling genes decreased with chilling, none of the protein levels showed a significant decrease, pointing at a differential regulation at the gene and protein level. ispartof: POSTHARVEST BIOLOGY AND TECHNOLOGY vol:155 pages:1-10 status: published

On average a human cell type expresses around 10,000 different protein coding genes synthesizing all the different molecular forms of the protein product (proteoforms) found in a cell. In a typical shotgun bottom up proteomic approach, the proteins are enzymatically cleaved, producing several 100,000 s of different peptides that are analyzed with liquid chromatography-tandem mass spectrometry (LC-MSMS). One of the major consequences of this high sample complexity is that coelution of peptides cannot be avoided. Moreover, low abundant peptides are difficult to identify as they have a lower chance of being selected for fragmentation due to ion-suppression effects and the semi-stochastic nature of the precursor selection in data-dependent shotgun proteomic analysis where peptides are selected for fragmentation analysis one-by-one as they elute from the column. In the current study we explore a simple novel approach that has the potential to counter some of the effect of coelution of peptides and improves the number of peptide identifications in a bottom-up proteomic analysis. In this method, peptides from a HeLa cell digest were eluted from the reverse phase column using three different elution solvents (acetonitrile, methanol and acetone) in three replicate reversed phase LC-MS/MS shotgun proteomic analysis. Results were compared with three technical replicates using the same solvent, which is common practice in proteomic analysis. In total, we see an increase of up to 10% in unique protein and up to 30% in unique peptide identifications from the combined analysis using different elution solvents when compared to the combined identifications from the three replicates of the same solvent. In addition, the overlap of unique peptide identifications common in all three LC-MS analyses in our approach is only 23% compared to 50% in the replicates using the same solvent. The method presented here thus provides an easy to implement method to significantly reduce the effects of coelution and ion suppression of peptides and improve protein coverage in shotgun proteomics. Data are available via ProteomeXchange with identifier PXD011908 .

Traditionally, immunohistochemistry (IHC) is used by pathologists to localise specific proteins or peptides in tissue slides. In the era of personalised medicine, however, molecular tissue analysis becomes indispensable for correct diagnosis, prognosis and therapeutic decision, not only on the DNA or mRNA level but also on the protein level. Combining molecular information with imaging presents many advantages. Therefore, matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) is a promising technique to be added to the armamentarium of the pathologist. Here, we focus on the workflow, advantages and drawbacks of both MALDI IMS and IHC. We also briefly discuss a few other protein imaging modalities and give examples of applications.

Maternal lipolytic metabolic disorders result in a lipotoxic microenvironment in the ovarian follicular fluid (FF) which deteriorates oocyte quality. Although cellular stress response mechanisms are well defined in somatic cells, they remain largely unexplored in oocytes, which have distinct organelle structure and nuclear transcription patterns. Here we used shotgun proteomic analyses to study cellular responses of bovine oocytes and cumulus cells (CCs) after in vitro maturation under lipotoxic conditions; in the presence of pathophysiological palmitic acid (PA) concentration as a model. Differentially regulated proteins (DRPs) were mainly localized in the endoplasmic reticulum, mitochondria and nuclei of CCs and oocytes, however the DRPs and their direction of change were cell-type specific. Proteomic changes in PA-exposed CCs were predominantly pro-apoptotic unfolded protein responses (UPRs), mitochondrial and metabolic dysfunctions, and apoptotic pathways. This was also functionally confirmed. Interestingly, although the oocytes were enclosed by CCs during PA exposure, elevated cellular stress levels were also evident. However, pro-survival UPRs, redox regulatory and compensatory metabolic mechanisms were prominent despite evidence of mitochondrial dysfunction, oxidative stress, and reduced subsequent embryo development. The data provides a unique insight that enriches the understanding of the cellular stress responses in metabolically-compromised oocytes and forms a fundamental base to identify new targets for fertility treatments as discussed within.

Transcriptome and ribosome sequencing have revealed the existence of many non-canonical transcripts, mainly containing splice variants, ncRNA, sORFs and altORFs. However, identification and characterization of products that may be translated out of these remains a challenge. Addressing this, we here report on 552 non-canonical proteins and splice variants in the model organism C. elegans using tandem mass spectrometry. Aided by sequencing-based prediction, we generated a custom proteome database tailored to search for non-canonical translation products of C. elegans. Using this database, we mined available mass spectrometric resources of C. elegans, from which 51 novel, non-canonical proteins could be identified. Furthermore, we utilized diverse proteomic and peptidomic strategies to detect 40 novel non-canonical proteins in C. elegans by LC-TIMS-MS/MS, of which 6 were common with our meta-analysis of existing resources. Together, this permits us to provide a resource with detailed annotation of 467 splice variants and 85 novel proteins mapped onto UTRs, non-coding regions and alternative open reading frames of the C. elegans genome.

To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery. ispartof: PLANT CELL AND ENVIRONMENT vol:43 issue:9 pages:2254-2271 ispartof: location:United States status: published

Studying the proteome–the entire set of proteins in cells, tissues, organs and body fluids—is of great relevance in cancer research, as differential forms of proteins are expressed in response to specific intrinsic and extrinsic signals. Discovering protein signatures/pathways responsible for cancer transformation may lead to a better understanding of tumor biology and to a more effective diagnosis, prognosis, recurrence and response to therapy. Moreover, proteins can act as a biomarker or potential drug targets. Hence, it is of major importance to implement proteomic, particularly mass spectrometric, approaches in cancer research, to provide new crucial insights into tumor biology. Recently, mass spectrometry imaging (MSI) approaches were implemented in cancer research, to provide individual molecular characteristics of each individual tumor while retaining molecular spatial distribution, essential in the context of personalized disease management and medicine.

Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.

The salivary protein repertoire released by the herbivorous pest Tetranychus urticae is assumed to hold keys to its success on diverse crops. We report on a spider mite–specific protein family that is expanded in T. urticae. The encoding genes have an expression pattern restricted to the anterior podocephalic glands, while peptide fragments were found in the T. urticae secretome, supporting the salivary nature of these proteins. As peptide fragments were identified in a host-dependent manner, we designated this family as the SHOT (secreted host–responsive protein of Tetranychidae) family. The proteins were divided in three groups based on sequence similarity. Unlike TuSHOT3 genes, TuSHOT1 and TuSHOT2 genes were highly expressed when feeding on a subset of family Fabaceae, while expression was depleted on other hosts. TuSHOT1 and TuSHOT2 expression was induced within 24 h after certain host transfers, pointing toward transcriptional plasticity rather than selection as the cause. Transfer from an ‘inducer’ to a ‘noninducer’ plant was associated with slow yet strong downregulation of TuSHOT1 and TuSHOT2, occurring over generations rather than hours. This asymmetric on and off regulation points toward host-specific effects of SHOT proteins, which is further supported by the diversity of SHOT genes identified in Tetranychidae with a distinct host repertoire.

The two-spotted spider mite Tetranychus urticae is an extremely polyphagous crop pest. Alongside an unparalleled detoxification potential for plant secondary metabolites, it has recently been shown that spider mites can attenuate or even suppress plant defenses. Salivary constituents, notably effectors, have been proposed to play an important role in manipulating plant defenses and might determine the outcome of plant-mite interactions. Here, the proteomic composition of saliva from T. urticae lines adapted to various host plants—bean, maize, soy, and tomato—was analyzed using a custom-developed feeding assay coupled with nano-LC tandem mass spectrometry. About 90 putative T. urticae salivary proteins were identified. Many are of unknown function, and in numerous cases belonging to multimembered gene families. RNAseq expression analysis revealed that many genes coding for these salivary proteins were highly expressed in the proterosoma, the mite body region that includes the salivary glands. A subset of genes encoding putative salivary proteins was selected for whole-mount in situ hybridization, and were found to be expressed in the anterior and dorsal podocephalic glands. Strikingly, host plant dependent expression was evident for putative salivary proteins, and was further studied in detail by micro-array based genome-wide expression profiling. This meta-analysis revealed for the first time the salivary protein repertoire of a phytophagous chelicerate. The availability of this salivary proteome will assist in unraveling the molecular interface between phytophagous mites and their host plants, and may ultimately facilitate the development of mite-resistant crops. Furthermore, the technique used in this study is a time- and resource-efficient method to examine the salivary protein composition of other small arthropods for which saliva or salivary glands cannot be isolated easily.

© 2019 Van de Walle et al., The onset of sexual maturity involves dramatic changes in physiology and gene expression in many animals. These include abundant yolk protein production in egg-laying species, an energetically costly process under extensive transcriptional control. Here, we used the model organism Caenorhabditis elegans to provide evidence for the spatiotemporally defined interaction of two evolutionarily conserved transcription factors, CEH-60/PBX and UNC-62/MEIS, acting as a gateway to yolk protein production. Via proteomics, bimolecular fluorescence complementation (BiFC), and biochemical and functional readouts, we show that this interaction occurs in the intestine of animals at the onset of sexual maturity and suffices to support the reproductive program. Our electron micrographs and functional assays provide evidence that intestinal PBX/MEIS cooperation drives another process that depends on lipid mobilization: the formation of an impermeable epicuticle. Without this lipid-rich protective layer, mutant animals are hypersensitive to exogenous oxidative stress and are poor partners for mating. Dedicated communication between the hypodermis and intestine in C. elegans likely supports these physiological outcomes, and we propose a fundamental role for the conserved PBX/MEIS interaction in multicellular signaling networks that rely on lipid homeostasis., PVdW is an SB PhD fellow of the FWO Flanders. The authors are grateful to the FWO Flanders (G095915) and KU Leuven for funding.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane protein receptors. These receptors regulate a wide array of physiological processes in both healthy and diseased conditions via G protein-dependent and –independent signaling pathways. Remarkably, a subfamily of class A GPCRs, Mas-related G protein-coupled receptors (MRGPRs), is predominantly expressed in sensory neurons of the dorsal root and trigeminal ganglia, as well as on mast cells. Interestingly, we recently discovered that Mas-related G protein-coupled receptor F (MRGPRF), which is known to be expressed by brain, spinal cord and enteric neurons, is also expressed by enteroendocrine cells in the gastrointestinal tract. MRGPRF expression was predominantly found at basal and basolateral sites, indicating a novel role in endocrine regulation. However, due to the orphan nature of MRGPRF, little is known about its role in the pathophysiology of intestinal inflammation. Additionally, increasing evidence suggests a prominent role of peptides and proteases, predominantly secreted by macrophages, microglia and enteroendocrine cells, in inflammatory conditions of the gut, but the therapeutic perspectives are still restricted because the underlying molecular mechanisms are unknown. Here, we report for the first time on the activation of human MRGPRF by the cysteine protease Cathepsin S (Cat-S). To this end, we first performed in-silico analysis of the N-terminal sequence of MRGPRF, which revealed consensus motif sites of cleavage by Cat-S. Furthermore, the N-terminal peptide of MRGPRF was synthesized, incubated with Cat-S and analyzed for cleaved sites using mass spectroscopy. The results clearly showed cleavage sites and as such corroborated our in-silico analysis. Moreover, luciferase tagged at the N-terminus of MRGPRF was expressed in HeLa cells incubated with Cat-S and receptor N-terminal cleavage was assessed by measuring luminescence activity in the supernatant. The high luciferase activity in the supernatant compared to its control (no Cat-S treatment) indicated cleavage of the N-terminus of MRGPRF. We next sought to determine receptor activation upon Cat-S treatment using live cell calcium imaging with Fluo-4. HeLa cells transiently transfected with MRGPRF and incubated with Cat-S, gave rise to protease-based receptor activation, as evident by a fluorescence trace when compared to controls (non-transfected HeLa cells). These results pave the way for deorphanization of MRGPRF and call for further pathological investigations related to MRGPRF, in particular with regard to its involvement in neuro-immune communication within the gut.

In the context of omics disciplines and especially proteomics and biomarker discovery, the analysis of a clinical sample using label-based tandem mass spectrometry (MS) can be affected by sample preparation effects or by the measurement process itself, resulting in an incorrect outcome. Detection and correction of these mistakes using state-of-the-art methods based on, f.i., mixed models can take a lot of (computing) time. MS-based proteomics labs are high-throughput and need to avoid a bottleneck in their quantitative pipeline by quickly discriminating between high- and low-quality data. To this end we developed an easy-to-use web-tool called QCQuan (available at https://qcquan.net/) which is built around the CONSTANd normalization algorithm. It automatically provides the user with exploratory and quality control information as well as a differential expression analysis based on conservative, simple statistics. In this document we describe in detail the scientifically relevant steps that constitute the workflow, and assess its qualitative and quantitative performance on three reference data sets. We find that QCQuan provides clear and accurate indications about the scientific value of both a high- and a low-quality data set. Moreover, it performed quantitatively better on a third data set than a comparable workflow assembled using established, reliable software.

Naturally occurring peptides, including growth factors, hormones, and neurotransmitters, represent an important class of biomolecules and have crucial roles in human physiology. The study of these peptides in clinical samples is therefore as relevant as ever. Compared to more routine proteomics applications in clinical research, peptidomics research questions are more challenging and have special requirements with regard to sample handling, experimental design, and bioinformatics. In this review, we describe the issues that confront peptidomics in a clinical context. After these hurdles are (partially) overcome, peptidomics will be ready for a successful translation into medical practice.

Neuroglobin is a heme protein of which increased levels provide neuroprotection against amyloid proteinopathy and hemorrhagic damage. These cellular stressors involve the promotion of ferroptosis&mdash, an iron-dependent, lipid peroxide-accreting form of cell death. Hence, we questioned whether neuroglobin could oppose ferroptosis initiation. We detected human neuroglobin (hNgb)-EGFP-expressing SH-SY5Y cells to be significantly more resistant to ferroptosis induction, identifying 0.68-fold less cell death. To elucidate the underlying pathways, this study investigated hNgb-protein interactions with a Co-IP-MS/MS approach both under a physiological and a ferroptotic condition. hNgb binds to proteins of the cellular iron metabolism (e.g., RPL15 and PCBP3) in an unstressed condition and shows an elevated binding ratio towards cell death-linked proteins, such as HNRNPA3, FAM120A, and ABRAXAS2, under ferroptotic stress. Our data also reveal a constitutive interaction between hNgb and the longevity-associated heterodimer XRCC5/XRCC6. Disentangling the involvement of hNgb and its binding partners in cellular processes, using Ingenuity Pathway Analysis, resulted in the integration of hNgb in the ubiquitination pathway, mTOR signaling, 14-3-3-mediated signaling, and the glycolysis cascade. We also detected a previously unknown strong link with motor neuropathies. Hence, this study contributes to the elucidation of neuroglobin&rsquo, s involvement in cellular mechanisms that provide neuroprotection and the upkeep of homeostasis.

Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs.

Simple Summary The introduction of immunotherapy modified the cancer treatment landscape, especially for non-small cell lung cancer (NSCLC). Unfortunately, only a subgroup of patients benefits from this therapy. Currently, the only validated companion diagnostic test for first-line immunotherapy in metastatic NSCLC patients is testing for programmed death ligand 1 (PD-L1) expression in tumor tissues. However, obtaining tumor tissue can be challenging and it puts the patient at risk. Liquid biopsy offers an alternative, less invasive approach to select NSCLC patients who would benefit from immunotherapy and to monitor patients during their disease course. Liquid biopsy allows repetitive sampling, which makes it a useful tool in clinical practice. In this review, we discuss the challenges and opportunities of several liquid biopsy-based prognostic and predictive biomarkers in NSCLC patients receiving immunotherapy. In the last decade, immunotherapy has been one of the most important advances in the non-small cell lung cancer (NSCLC) treatment landscape. Nevertheless, only a subset of NSCLC patients benefits from it. Currently, the only Food and Drug Administration (FDA) approved diagnostic test for first-line immunotherapy in metastatic NSCLC patients uses tissue biopsies to determine the programmed death ligand 1 (PD-L1) status. However, obtaining tumor tissue is not always feasible and puts the patient at risk. Liquid biopsy, which refers to the tumor-derived material present in body fluids, offers an alternative approach. This less invasive technique gives real-time information on the tumor characteristics. This review addresses different promising liquid biopsy based biomarkers in NSCLC patients that enable the selection of patients who benefit from immunotherapy and the monitoring of patients during this therapy. The challenges and the opportunities of blood-based biomarkers such as cell-free DNA (cfDNA), circulating tumor cells (CTCs), exosomes, epigenetic signatures, microRNAs (miRNAs) and the T cell repertoire will be addressed. This review also focuses on the less-studied feces-based and breath-based biomarkers.