Background Glanzmann thrombasthenia (GT) is an inherited platelet function disorder caused by mutations in the fibrinogen receptor αIIbβ3. The deficiency can be quantitative (type I/II) or qualitative (type III). It causes lack of platelet aggregation and leads to a moderate to severe bleeding tendency. Besides the absence or functional alteration of the integrins αIIb and β3, little is known about the proteomic landscape of platelets from GT patients. Objectives To evaluate the platelet proteome in GT. Methods Label-free quantification of platelet proteins was performed in 13 genetically confirmed GT patients (11 type I and 2 type III) and 13 healthy controls with liquid chromatography coupled with tandem mass spectrometry. αIIbβ3 expression was quantified with whole blood flow cytometry. Medical ethics committee approval was obtained and all participants provided informed consent. Results Of 3664 identified proteins, 2677 were considered quantified. Dynamic range spanned 5 orders of magnitude, and the mean coefficient of variation was 1.2%, indicating data were robust. Flow cytometry-based αIIb expression correlated well with αIIb abundance according to liquid chromatography-tandem mass spectrometry. Twenty-nine proteins were less abundant, and 32 proteins were more abundant in GT patients than in controls. Downregulated proteins were enriched for α-granule proteins, including secreted protein acidic and cyteine rich, amyloid β precursor-like protein 2, TIMP metallopeptidase inhibitor 1, and TREM-like transcript-1, in addition to the subunits of integrin αIIbβ3, fibrinogen, and plasminogen. Upregulated proteins were mostly plasma proteins annotated to blood microparticles. Conclusion GT platelets show reduced abundance of specific platelet α-granule proteins compared with healthy controls.

Aims In this study, we aimed to uncover genes associated with stressed cardiomyocytes by combining single-cell transcriptomic data sets from failing cardiac tissue from both humans and mice.Methods and results Our bioinformatic analysis identified SORBS2 as conserved NPPA-correlated gene. Using mouse models and cardiac tissue from human heart failure patients, we demonstrated that SORBS2 expression is consistently increased during pathological remodelling, correlates to disease severity, and is regulated by GATA4. By affinity purification mass spectrometry, we showed SORBS2 to interact with the integrin-cytoskeleton connections. Cardiomyocyte-specific genetic loss of Sorbs2 in adult mice changed integrin interactions, indicated by the increased expression of several integrins and altered extracellular matrix components connecting to these integrins, leading to an exacerbated fibrotic response during pathological remodelling.Conclusion Sorbs2 is a cardiomyocyte-enriched gene that is increased during progression to heart failure in a GATA4-dependent manner and correlates to phenotypical hallmarks of cardiac failure. Our data indicate SORBS2 to function as a crucial regulator of integrin interactions and cardiac fibrosis.

Lysosomes are membrane-bound organelles critical for maintaining cellular homeostasis. Delivery of biosynthetic lysosomal proteins to lysosomes is crucial to orchestrate proper lysosomal function. However, it remains unknown how the delivery of biosynthetic lysosomal proteins to lysosomes is ensured in neurons, which are highly polarized cells. Here, we developed Protein Origin, Trafficking And Targeting to Organelle Mapping (POTATOMap), by combining trafficking synchronization and proximity-labelling based proteomics, to unravel the trafficking routes and interactome of the biosynthetic lysosomal membrane protein LAMP1 at specified time points. This approach, combined with advanced microscopy, enables us to identify the neuronal domain-specific trafficking machineries of biosynthetic LAMP1. We reveal a role in replenishing axonal lysosomes, in delivery of newly synthesized axonal synaptic proteins, and interactions with RNA granules to facilitate hitchhiking in the axon. POTATOMap offers a robust approach to map out dynamic biosynthetic protein trafficking and interactome from their origin to destination.

The neuronal cell adhesion molecule contactin-4 (CNTN4) is genetically associated with autism spectrum disorder (ASD) and other psychiatric disorders. Cntn4-deficient mouse models have previously shown that CNTN4 plays important roles in axon guidance and synaptic plasticity in the hippocampus. However, the pathogenesis and functional role of CNTN4 in the cortex has not yet been investigated. Our study found a reduction in cortical thickness in the motor cortex of Cntn4 −/− mice, but cortical cell migration and differentiation were unaffected. Significant morphological changes were observed in neurons in the M1 region of the motor cortex, indicating that CNTN4 is also involved in the morphology and spine density of neurons in the motor cortex. Furthermore, mass spectrometry analysis identified an interaction partner for CNTN4, confirming an interaction between CNTN4 and amyloid-precursor protein (APP). Knockout human cells for CNTN4 and/or APP revealed a relationship between CNTN4 and APP. This study demonstrates that CNTN4 contributes to cortical development and that binding and interplay with APP controls neural elongation. This is an important finding for understanding the physiological function of APP, a key protein for Alzheimer’s disease. The binding between CNTN4 and APP, which is involved in neurodevelopment, is essential for healthy nerve outgrowth.

Aspergillus niger is widely used as a cell factory for the industrial production of enzymes. Previously, it was shown that deletion of α-1–3 glucan synthase genes results in smaller micro-colonies in liquid cultures of Aspergillus nidulans. Also, it has been shown that small wild-type Aspergillus niger micro-colonies secrete more protein than large mirco-colonies. We here assessed whether deletion of the agsC or agsE α-1–3 glucan synthase genes results in smaller A. niger micro-colonies and whether this is accompanied by a change in protein secretion. Biomass formation was not affected in the deletion strains but pH of the culture medium had changed from 5.2 in the case of the wild-type to 4.6 and 6.4 for ΔagsC and ΔagsE, respectively. The diameter of the ΔagsC micro-colonies was not affected in liquid cultures. In contrast, diameter of the ΔagsE micro-colonies was reduced from 3304 ± 338 µm to 1229 ± 113 µm. Moreover, the ΔagsE secretome was affected with 54 and 36 unique proteins with a predicted signal peptide in the culture medium of MA234.1 and the ΔagsE, respectively. Results show that these strains have complementary cellulase activity and thus may have complementary activity on plant biomass degradation. Together, α-1–3 glucan synthesis (in)directly impacts protein secretion in A. niger.

Aspergillus niger can form microcolonies of different size due to partial aggregation of spores and germlings. So far, this heterogeneity was considered a negative trait by the industry.

G-quadruplex (or G4) structures form in guanine- rich DNA sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that acts during DNA replication. First, the replicative helicase composed of Cdc45, MCM2-7 and GINS (CMG) stalls at a leading strand G4 structure. Second, the DEAH-box helicase 36 (DHX36) mediates bypass of the CMG past the intact G4 structure, allowing approach of the leading strand to the G4. Third, G4 structure unwinding by the Fanconi anemia complementation group J helicase (FANCJ) enables DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG but still requires DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring pathway robustness. This previously unknown genome maintenance pathway promotes faithful G4 replication, thereby avoiding genome instability.

While endocrine therapy is highly effective for the treatment of oestrogen receptor-α (ERα)-positive breast cancer, a significant number of patients will eventually experience disease progression and develop treatment-resistant, metastatic cancer. The majority of resistant tumours remain dependent on ERα-action, with activating ESR1 gene mutations occurring in 15–40% of advanced cancers. Therefore, there is an urgent need to discover novel effective therapies that can eradicate cancer cells with aberrant ERα and to understand the cellular response underlying their action. Here, we evaluate the response of MCF7-derived, CRISPR-Cas9-generated cell lines expressing mutant ERα (Y537S) to a large number of drugs. We report sensitivity to numerous clinically approved inhibitors, including CDK4/6 inhibitor ribociclib, which is a standard-of-care therapy in the treatment of metastatic ERα-positive breast cancer and currently under evaluation in the neoadjuvant setting. Ribociclib treatment induces senescence in both wildtype and mutant ERα breast cancer models and leads to a broad-range drug tolerance. Strikingly, viability of cells undergoing ribociclib-induced cellular senescence is maintained via engagement of EGFR signalling, which may be therapeutically exploited in both wildtype and mutant ERα-positive breast cancer. Our study highlights a wide-spread reduction in sensitivity to anti-cancer drugs accompanied with an acquired vulnerability to EGFR inhibitors following CDK4/6 inhibitor treatment.

Acquired resistance to MAPK inhibitors limits the clinical efficacy in melanoma treatment. We and others have recently shown that BRAF inhibitor (BRAFi)-resistant melanoma cells can develop a dependency on the therapeutic drugs to which they have acquired resistance, creating a vulnerability for these cells that can potentially be exploited in cancer treatment. In drug-addicted melanoma cells, it was shown that this induction of cell death was preceded by a specific ERK2-dependent phenotype switch; however, the underlying molecular mechanisms are largely lacking. To increase the molecular understanding of this drug dependency, we applied a mass spectrometry-based proteomic approach on BRAFi-resistant BRAFMUT 451Lu cells, in which ERK1, ERK2, and JUNB were silenced separately using CRISPR-Cas9. Inactivation of ERK2 and, to a lesser extent, JUNB prevents drug addiction in these melanoma cells, while, conversely, knockout of ERK1 fails to reverse this phenotype, showing a response similar to that of control cells. Our analysis reveals that ERK2 and JUNB share comparable proteome responses dominated by reactivation of cell division. Importantly, we find that EMT activation in drug-addicted melanoma cells upon drug withdrawal is affected by silencing ERK2 but not ERK1. Moreover, transcription factor (regulator) enrichment shows that PIR acts as an effector of ERK2 and phosphoproteome analysis reveals that silencing of ERK2 but not ERK1 leads to amplification of GSK3 kinase activity. Our results depict possible mechanisms of drug addiction in melanoma, which may provide a guide for therapeutic strategies in drug-resistant melanoma.

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.

Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene flbA results in mycelial colonies that secrete proteins throughout the colony. However, ΔflbA strain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which, rpnR, was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the ΔrpnR deletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when A. niger was grown on xylose. Moreover, the ΔrpnR strain showed decreased resistance to H2O2 and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when A. niger is grown on xylose.IMPORTANCEAspergillus niger secretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of A. niger are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in A. niger by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production in A. niger.

Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set ( n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC.

The coprophilic ascomycete fungus Podospora anserina was cultivated on three different plant biomasses, i.e. cotton seed hulls (CSH), soybean hulls (SBH) and acid-pretreated wheat straw (WS) for four days, and the potential of the produced enzyme mixtures was compared in the enzymatic saccharification of the corresponding lignocellulose feedstocks. The enzyme cocktail P. anserina produced after three days of growth on SBH showed superior capacity to release reducing sugars from all tested plant biomass feedstocks compared to the enzyme mixtures from CSH and WS cultures. Detailed proteomics analysis of the culture supernatants revealed that SBH contained the most diverse set of enzymes targeted on plant cell wall polymers and was particularly abundant in xylan, mannan and pectin acting enzymes. The importance of lytic polysaccharide monooxygenases (LPMOs) in plant biomass deconstruction was supported by identification of 20 out of 33 AA9 LPMOs in the SBH cultures. The results highlight the suitability of P. anserina as a source of plant cell wall degrading enzymes for biotechnological applications and the importance of selecting the most optimal substrate for the production of enzyme mixtures.

Neospora caninum is an obligatory intracellular Apicomplexan protozoan parasite that must invade cells for its development. Thrombospondin-related proteins are Acomponents of the parasite micronemes, organelles that have an important role in Apicomplexa adhesion and invasion and are currently promising targets for malaria vaccine research. In Neospora caninum, two thrombospondin-related protein homologues have been described: NcMIC2 and NcMIC2-like1. This work extends the information about microneme proteins in N. caninum by employing mass spectrometry data on tachyzoite proteins. The proteins were isolated by two-dimensional SDS-PAGE, uncovering some details not described in previous works that were based on one-dimensional SDS-PAGE. The compilation of NcMIC2-like1 and NcMIC2 data in a unique approach will guide future studies related to the blockage and manipulation of these proteins, improving the alternatives for neosporosis prevention. Introduction Neosporosis is a disease related to economic losses in livestock industry due to fetal abortion, stillbirth, clinical and subclinical diseases, impaired milk production, neonatal deaths, and reduced fertility [1]. Neospora caninum is an obligate intracellular parasite that invades host cells by a conserved mechanism, which is typical to the Apicomplexa phylum [2] [3]. The invasion process is active, parasite-coordinated and multifactorial, involving the release of proteins from organelles such as micronemes, rhoptries, and dense granules [4]. Apicomplexans use a substrate locomotion process called gliding motility that involves both the parasite cytoskeleton and the host cell [5]. In this context, the thrombospondin-related anonymous proteins (TRAP or MIC2 proteins) are considered key elements, since they interact simultaneously with host cell receptors and the actin-myosin motor of the parasite. TRAPs or MIC2 proteins in Apicomplexan parasites have two types of extracellular adhesive domains, one vonWillebrand factor type A domain (or integrin IA) and at least one Thrombospondin type 1 domain (TSP-1). There are also a signal peptide, a transmembrane region with a rhomboid cleavage site and an acidic cytoplasmic tail with a conserved tryptophan residue close to the C-terminal end [4]. TRAP family members include Plasmodium falciparum (PfTRAP), Toxoplasma gondii (TgMIC2) [6], and N. caninum (NcMIC 2 and NcMIC2-like1) homologues [7] [8]. The total disruption of Tgmic2 confirmed the role of this protein in parasite replication and growth [9]. Based on the relevance of Apicomplexan TRAP proteins and their potential in the life cycle of N. caninum, our research group is interested in the molecular characterization of NcMIC2-like1 [8]. The present work includes a proteomic study of MIC2 proteins of N. caninum (NcMIC2-like1 and NcMIC2), improving the data about this protein family, closely related to the parasite invasion mechanism. Objective The aim of this work was to detect and identify TRAP proteins from N. caninum (NcMIC2-like1 and NcMIC2) using 2D SDS-PAGE and mass spectrometry. Proteomic data on thrombospondin-related proteins (TRAP) from Neospora caninum (NcMIC2-like1 and NcMIC2) DOI: 10.19185/matters.201702000002 Matters (ISSN: 2297-8240) | 2 a Proteomic data on thrombospondin-related proteins (TRAP) from Neospora caninum (NcMIC2-like1 and NcMIC2) DOI: 10.19185/matters.201702000002 Matters (ISSN: 2297-8240) | 3 Figure Legend Figure 1. Proteomic detection ofN. caninum TRAP proteins (NcMIC2-like1 and NcMIC2). (A) NcMIC2-like1 and NcMIC2 were detected in the ESA and N. caninum tachyzoite extracts by shotgun proteomics according to Pollo-Oliveira et al. (2013). (B) Acidic forms of NcMIC2-like1 and NcMIC2 localized on a two-dimensional coomassie gel (10%, pH 3–5.6, non-linear, 7 cm). The spots (indicated by numbers 1, 2, 3, and 4) were identified as NcMIC2-like1 (spots 1 and 2) and NcMIC2 (spots 3 and 4). (C)Detection of native NcMIC2-like1 (spots 1 and 2) by 2DWestern blot (10%, pH 3–5.6, non-linear, 7 cm) using anti-recombinant NcMIC2-like1 serum onN. caninum tachyzoite protein extract. (D) Isoelectric point (pI), molecular weight (MW), and number of unique peptides and peptide spectrum matches of spots 1, 2, 3, and 4 after MS/MS analysis. Tissue culture and parasite purification Vero cell cultures were maintained in RPMI-1640 medium (Sigma) supplemented with 5% fetal calf serum (Cultilab), 2.05 mM glutamine, 50 U penicillin/streptomycin in T-25 or 75 tissue culture flasks at 37°C and 5% CO2. The cultures were trypsinized at least once a week. N. caninum tachyzoites from the Nc-1 isolate were maintained in Vero cell monolayers and purified by exclusion chromatography in Sephadex G-25 (PD-10 columns; GE). Data analysis TheN. caninum data from high-resolutionmass spectrometry-based proteomics was obtained as previously described [10]. Proteins present in the ESA and in the discharged tachyzoites (parasites submitted to an ethanol secretion stimulus) were identified using shotgun LC-MS/MS. The protein IDs were generated after separation of the N. caninum proteins by strong cation exchange chromatography. From this work, 615 proteins were identified in ESA and 2,011 proteins quantified in the discharged tachyzoites; however, few properties about MIC2 proteins family were deeply analysed. Therefore, in this article, all the PSMs were analyzed in detail for NcMIC2-like 1 and NcMIC2 identification/quantification. 2D SDS-PAGE The N. caninum tachyzoite protein extract, prepared as described previously [8], was dissolved (50 μg) in a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% carrier ampholyte mixture (pH 3–5.6 NL in Immobiline Drystrip gels; GE), 20 mM dithiothreitol and supplemented with protease inhibitors (1/100, Protease Inhibitor cocktail; Sigma-Aldrich). The samples were loaded on 7 cm IPG strips (pH 3–5.6 NL), rehydrated and focused in an automated overnight run (IPGPhor) using 10–14 h of rehydration followed by a step voltage focusing procedure (20 min 100 V, 30 min 300 V, 1.2 h 1,000 V, 15 min 5,000 V, followed by 5,000 V until a total of 5 kVh was reached). After focusing, the strips were incubated with 30 mM dithiothreitol and 135 mM iodoacetamide in the equilibration buffer (50 mM Tris, 6 M urea, 2% SDS, 30% glycerol, pH 8.8) for 15 min. The proteins were separated by 10% SDS-PAGE and the gels stained with Coomassie Brilliant Blue G 250. Gel images were acquired with LabScan v5.0 software on a flatbed scanner (Image Scanner; GE). Western blot The 2D gels of N. caninum tachyzoite protein extracts were transferred to a PVDF membrane (Immobilon-P; Millipore) with a semi-dry system (TE 77 PWR; Amersham Biosciences) using transfer buffer (39 mM glycine, 48 mM Tris, 0.0375% SDS, 20% methanol) at 0.81 mA/cm2 for 1.15 h. The blot was blocked with 0.8% porcine gelatin (SigmaAldrich) diluted in phosphate-buffered saline containing 0.05% Tween (PBST) for 1 h at 37°C. The antiserum anti-NcMIC2-like1 [8] was diluted (1/2,000) in 0.2% porcine gelatin PBST and incubated overnight at 4°C. After washing (three times with PBST), the blot was incubated with anti-rabbit-immunoglobulin conjugated to HRP (1/10,000; ZyMedInvitrogen) for 1 h at 37°C and then washed three times with PBST. The signal was obtained with an enhanced chemiluminescent substrate for HRP detection (SuperSignal West Pico Chemiluminescent Substrate; Pierce). The blots were scanned using the Image Scanner. Mass spectrometry Proteomic data on thrombospondin-related proteins (TRAP) from Neospora caninum (NcMIC2-like1 and NcMIC2) DOI: 10.19185/matters.201702000002 Matters (ISSN: 2297-8240) | 4 The spots were washed once with milliQ water and twice with 50 mM ammonium bicarbonate (pH 8.0) followed by shrinkage with 100% acetonitrile (ACN). For protein digestion, the spot was incubated overnight with trypsin (Promega) at 37°C. The peptides were extracted with 100% ACN, dried in a SpeedVac vacuum concentrator, and analyzed by RP-nano-LC-MS/MS. After resuspension in 10% formic acid, the peptides were loaded into a LTQ Orbitrap XL ETD mass spectrometer (Thermo Electron, Bremen, Germany) coupled to an Agilent 1200 HPLC system (Agilent Technologies), using a 20×100 mm trap column (Aquat C18, 5 mm; Phenomenex, Torrance, CA) and a 40 cm × 50 mm analytical column (ReproSil-Pur C18-AQ, 3 mm; Dr Maisch GmbH, Ammerbuch, Germany). A 60 min run was carried out using elution gradients with solvent A (0.6% acetic acid) and with solvent B (0.6% acetic acid in 80% ACN) with a flow rate of 100 nL/min. The five most intense peptide ions were selected for fragmentation by collision-induced dissociation. The outputs were compared with data in the N. caninum predicted protein database (ToxoDB 28) using the Mascot software version 2.4.01 (Matrix Science). Carbamidomethylation of cysteine and oxidation of methionine were set as fixed and variable modifications, respectively. The database search was performed with a peptide tolerance of 50 ppm and product ion tolerance of 0.6 Da, allowing two missed cleavages. Results & Discussion A shotgun (LC-MS/MS) proteomic approach on N. caninum, both from N. caninum tachyzoite lysate and secreted fraction (ESA), enabled the identification of a massive number of proteins, including NcMIC2-like1 and NcMIC2 [10]. In total, 490 peptide spectrum matches (PSMs) from NcMIC2-like1 and 168 from NcMIC2 were obtained, with a considerably higher presence of NcMIC2-like1 than NcMIC2 in the ESA (448 and 126 PSMs, respectively) and the same number of PSMs (42) in the tachyzoite extract (Fig. 1A). Among all the 615 proteins identified in ESA [10], NcMIC2-like1 was the 21st most frequently identified and NcMIC2 ranked 93rd. Since NcMIC2-like1 and NcMIC2 are predicted to be acidic (pI 4.5 for both), 2D gels were run carrying tachyzoite extracts using narrow acidic strips of 3–5.6 NL (nonlinear). Mass spectrometry revealed spots 1 and 2 (Fig. 1B) as NcMIC2-like1, whereas spots 3 and 4 presente

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.

The white button mushroom Agaricus bisporus is one of the most widely produced edible fungus with a great economical value. Its commercial cultivation process is often performed on wheat straw and animal manure based compost that mainly contains lignocellulosic material as a source of carbon and nutrients for the mushroom production. As a large portion of compost carbohydrates are left unused in the current mushroom cultivation process, the aim of this work was to study wild-type A. bisporus strains for their potential to convert the components that are poorly utilized by the commercial strain A15. We therefore focused our analysis on the stages where the fungus is producing fruiting bodies. Growth profiling was used to identify A. bisporus strains with different abilities to use plant biomass derived polysaccharides, as well as to transport and metabolize the corresponding monomeric sugars. Six wild-type isolates with diverse growth profiles were compared for mushroom production to A15 strain in semi-commercial cultivation conditions. Transcriptome and proteome analyses of the three most interesting wild-type strains and A15 indicated that the unrelated A. bisporus strains degrade and convert plant biomass polymers in a highly similar manner. This was also supported by the chemical content of the compost during the mushroom production process. Our study therefore reveals a highly conserved physiology for unrelated strains of this species during growth in compost.

Hypothesis‐driven MS‐based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data‐independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high‐throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well‐informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM3) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3‐kinase mechanistic target of rapamycin (PI3K‐mTOR) and mitogen‐activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM3, allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K‐mTOR and MAPK phosphosites, gaining a so far unachieved in‐depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non‐small cell lung cancer.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

In view of important neurobiological functions of the cell adhesion molecule contactin-6 (Cntn6) that have emerged from studies on null-mutant mice and autism spectrum disorders patients, we set out to examine pathways underlying functions of Cntn6 using a proteomics approach. We identified the cell adhesion GPCR latrophilin-1 (Lphn1, a.k.a. CIRL1/CL, ADGRL1) as a binding partner for Cntn6 forming together a heteromeric cis-complex. Lphn1 expression in cultured neurons caused reduction in neurite outgrowth and increase in apoptosis, which was rescued by coexpression of Cntn6. In cultured neurons derived from Cntn6-/- mice, Lphn1 knockdown reduced apoptosis, suggesting that the observed apoptosis was Lphn1-dependent. In line with these data, the number of apoptotic cells was increased in the cortex of Cntn6-/- mice compared to wild-type littermate controls. These results show that Cntn6 can modulate the activity of Lphn1 by direct binding and suggests that Cntn6 may prevent apoptosis thereby impinging on neurodevelopment.