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The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell-free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.

Here the relation between digestion of proteins by trypsin covalently bound to paper and trypsin in‐solution is investigated. The trypsin acting on paper is covalently bound. A trypsin concentration of 0.5% (w/v) results in the highest digestion activity of all concentrations tested. Additionally, it can be seen that trypsin on‐paper has retained approx. 50% of its activity. Unlike trypsin in‐solution, the stability of the smart proteolysis samplers was regarded to be stable for at least four months when kept refrigerated. Autolysis was very small for covalently bound trypsin: less than 2% compared to in‐solution trypsin. Proteomic analysis of diluted human serum showed more protein identifications (214) in‐solution digestions than on‐paper digestions (76). Also, higher coverage for the in‐solution digestion was obtained. Those proteins identified after on‐paper digestion with no or few disulfide bonds seem to have more similar sequence coverages compared to those identified after in‐solution digestion. Smart samplers allow the determination of at least 70–75 proteins without performing the overnight digestion. All in all, trypsin covalently bound to paper shows to retain high proteolytic activity and is a stable alternative for conventional digestions. In this way, smart proteolytic samplers show their feasibility in pre‐lab sample preparation. [ABSTRACT FROM AUTHOR]

More than a decade has passed since the finalization of the Human Genome Project. Omics technologies made a huge leap from trendy and very expensive to routinely executed and relatively cheap assays. Simultaneously, we understood that omics is not a panacea for every problem in the area of human health and personalized medicine. Whilst in some areas of research omics showed immediate results, in other fields, including asthma, it only allowed us to identify the incredibly complicated molecular processes. Along with their possibilities, omics technologies also bring many issues connected to sample collection, analyses and interpretation. It is often impossible to separate the intrinsic imperfection of omics from asthma heterogeneity. Still, many insights and directions from applied omics were acquired-presumable phenotypic clusters of patients, plausible biomarkers and potential pathways involved. Omics technologies develop rapidly, bringing improvements also to asthma research. These improvements, together with our growing understanding of asthma subphenotypes and underlying cellular processes, will likely play a role in asthma management strategies., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Workplace‐collected blood spots deposited on filter paper were analysed with multiplexed affinity‐based protein assays and found to be suitable for proteomics analysis. The protein extension assay (PEA) was used to characterize 92 proteins using 1.2 mm punches in repeated samples collected from 20 workers. Overall, 97.8% of the samples and 91.3% of the analysed proteins passed quality control. Both within and between spot correlations using six replicates from the same individual were above 0.99, suggesting that comparable levels are obtained from multiple punches from the same spot and from consecutive spots. Protein levels from dried blood and wet serum from the same individuals were compared and the majority of the analysed proteins were found to be significantly correlated. These results open up for simplified sample collection of blood in field conditions for proteomic analysis, but also highlight that not all proteins can be robustly measured from dried whole blood. [ABSTRACT FROM AUTHOR]

Aim: Impairments in cerebral structure and cognitive performance in chronic heart failure (CHF) are critical components of its comorbidity spectrum. Autonomic afferents that arise from cardiac sensory fibres show enhanced activity with CHF. Desensitization of these fibres by local application of resiniferatoxin (RTX) during myocardial infarction (MI) is known to prevent cardiac hypertrophy, sympathetic hyperactivity and CHF. Whether these afferents mediate cerebral allostasis is unknown., Methods: CHF was induced by myocardial infarction. To evaluate if cardiac afferents contribute to cerebral allostasis, RTX was acutely applied to the pericardial space in controls (RTX) and in MI treated animals (MI/RTX). Subjects were then evaluated in a series of behavioural tests recapitulating different symptoms of depressive disorders. Proteomics of the frontal cortices (FC) was performed to identify contributing proteins and pathways responsible for behavioural allostasis., Results: Desensitization of cardiac afferents relieves hallmarks of an anxio/depressive-like state in mice. Unique protein signatures and regulatory pathways in FCs isolated from each treatment reveal the degree of complexity inherent in the FC response to stresses originating in the heart. While cortices from the combined treatment (MI/RTX) did not retain protein signatures from the individual treatment groups, all three groups suffer dysregulation in circadian entrainment., Conclusion: CHF is comorbid with an anxio/depressive-like state and ablation of cardiac afferents relieves the despair phenotype. The strikingly different proteomic profiles observed in FCs suggest that MI and RTX lead to unique brain-signalling patterns and that the combined treatment, potentially through destructive interference mechanisms, most closely resembles controls., (© 2020 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Quantitative translation of information on drug absorption, disposition, receptor engagement, and drug–drug interactions from bench to bedside requires models informed by physiological parameters that link in vitro studies to in vivo outcomes. To predict in vivo outcomes, biochemical data from experimental systems are routinely scaled using protein quantity in these systems and relevant tissues. Although several laboratories have generated useful quantitative proteomic data using state‐of‐the‐art mass spectrometry, no harmonized guidelines exit for sample analysis and data integration to in vivo translation practices. To address this gap, a workshop was held on September 27 and 28, 2018, in Cambridge, MA, with 100 experts attending from academia, the pharmaceutical industry, and regulators. Various aspects of quantitative proteomics and its applications in translational pharmacology were debated. A summary of discussions and best practices identified by this expert panel are presented in this "White Paper" alongside unresolved issues that were outlined for future debates. [ABSTRACT FROM AUTHOR]

We developed a curriculum for an upper‐level molecular biology course‐based undergraduate research laboratory class funded by a National Science Foundation CAREER grant that focuses on identifying new small proteins in the bacterium, Escherichia coli. Our CURE class has been continually offered each semester for the last 10 years, with multiple instructors collaboratively developing and implementing their own pedagogical approach while maintaining the same overall scientific goal and experimental strategy. In this paper, we delineate the experimental strategy for our molecular biology CURE laboratory class, describe a range of pedagogical approaches implemented by multiple instructors, and provide recommendations for teaching the class. The purpose of our paper is to share our experiences both in developing and teaching a molecular biology CURE laboratory class based on small protein identification and in creating a curriculum and support system that allows traditional, non‐traditional, and under‐represented students to participate in authentic research projects. [ABSTRACT FROM AUTHOR]

Introduction: Chromic myeloid leukemia (CML) blast crisis (BC) and imatinib (IM) resistance is a significant barrier to the effective treatment of the disease. Methods: Expression profiles of differential proteins were identified, and new biomarkers or pathways related to BC in CML were screened through proteomic analysis. Total proteins from primary bone marrow cells of CML patients in chronic phase (CP) and BC were separated via two-dimensional (2D) polyacrylamide gel electrophoresis and then analyzed by imagemaster 5.0 software to detect differential protein spots which were already identified by mass spectrometry. Based on the variation of the whole expression profile, some key proteins were picked out for Western blot to confirm the accuracy of proteomics data. Moreover, related signal pathways involving those proteins were investigated. Results: The result indicated that thirteen protein points between CML-CP and CML-BC were successfully determined. Results from Western blot of RhoA, hnRNPK, ANXA1, PSMB4, and LTA4H were similar to those from 2D polyacrylamide gel electrophoresis. Most of those proteins were involved in the proteosome pathway and the small G-protein pathway. Conclusion: A group of proteins associated with BC can be obtained and the result of this study might provide clues for further research. [ABSTRACT FROM AUTHOR]

Exercise can alter gene transcriptional and protein translational rates leading to changes in protein abundance toward adaptation to exercise. We investigated the alterations in protein abundance in skeletal muscle after one bout of an exhaustive exercise through proteomic analysis. Gastrocnemius muscles were sampled from non-exercised control rats and from rats exercised on a treadmill with incremental increases in speed until exhaustion (approximately 30 min). Rats were sacrificed 3 and 24 h after exercise cessation. Two-dimensional gel electrophoresis was performed and spots with a significant alteration in relative volume were identified by mass spectrometry. Six spots presented statistically significant altered abundances after exercise. The spots identified as the metabolic related proteins triosephosphate isomerase 1, glyceraldehyde-3-phosphate dehydrogenase, the β subunit of pyruvate dehydrogenase E1 and carnitine palmitoyltransferase 2 were all more abundant after exercise. One spot identified as heat shock cognate 70 was also more abundant after exercise. One spot demonstrated a decreased abundance after exercise and was identified as α-actin. These results suggest that a single session of exhaustive incremental exercise in untrained muscle can alter thin filaments synthesis/degradation rate and enhance cytosolic and mitochondrial proteins synthesis. The identified proteins may be important to a general preconditioning of skeletal muscle for subsequent exercise sessions. [ABSTRACT FROM AUTHOR]

Objectives: To use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein chip array technology to detect proteomic patterns in the serum of women with endometriosis; build diagnostic models; and evaluate their clinical significance.Methods: Serum samples from women with endometriosis and healthy women were studied using SELDI-TOF-MS protein chip technology. For every matched pair, two-thirds of the samples were used to look for different patterns and one-third was used for cross-validation.Results: Five potential biomarkers were found and the diagnostic system distinguished endometriosis from validation samples with a sensitivity of 91.7% and a specificity of 90.0%.Conclusion: This method shows great potential in identifying biomarkers to be used for endometriosis screening. [ABSTRACT FROM AUTHOR]

Summary: Localization of hot spots in a protein sequence is an important problem for understanding complex 3D structure of amino acid sequence (due to which biological functionality of protein is performed by their mutual interactions). Several digital signal processing (DSP) techniques like digital filtering, wavelet, and Fourier transforms have been used for finding the hot‐spot locations. Fast processing of these longer protein sequences is desirable. Field Programmable Gate Array (FPGA) implementation of Infinite Impulse Response (IIR) band pass notch (BPN) digital filter (tuned automatically by characteristics frequency of protein family) is carried out in this paper for further speed‐up of this protein analysis process. Performance of proposed filter system (implemented on Zynq series Zybo board FPGA) is further improved using retiming, which has significantly increased the maximum clock frequency from 34.304 to 54.812 MHz. Simulation of both MATLAB and FPGA hardware implementation for BPN IIR filter is executed on 10 protein functional groups. The simulation results predict the same hot‐spots locations as mentioned in ASEdb data base, but the Hardware BPN IIR filter (proposed in this paper) is 190 to 278 times faster as compared to its MATLAB counterpart. Some additional new hot‐spot locations (not mentioned in ASEdb data base) have been determined by our proposed hardware filter, which can be the probable hot‐spots for newly identified proteins. [ABSTRACT FROM AUTHOR]

Purpose: The management of blepharitis continues to challenge clinicians due to the poorly understood aetiology of the condition. We recently identified the family of intracellular plakin proteins as essential driving forces underlying anterior blepharitis. A large‐scale protein analysis was used to study if a topical dexamethasone/tobramycin solution could be used to reverse the expression of plakin proteins. Methods: Tear film samples from treatment naïve patients with anterior blepharitis (n = 15) were collected with Schirmer filtration paper. A subgroup of the patients (n = 10) received treatment with a dexamethasone/tobramycin 1 + 3 mg/mL ophthalmic suspension (Tobradex®) for 3 weeks and collection of tear film samples was repeated. The samples were analysed with label‐free quantification nano liquid chromatography–tandem mass spectrometry requiring quantification in at least 70% of the samples in each group. Proteins were considered differentially expressed if p < 0.05. Results: Following Tobradex® intervention, 27 proteins were upregulated while 61 proteins were downregulated. Regulated proteins after Tobradex® treatment were involved in intermediate filament cytoskeleton organization including downregulation of the plakin proteins envoplakin, epiplakin and periplakin. Plectin, a protein of the plakin family, remained unchanged after Tobradex® therapy. Tobradex® treatment resulted in the regulation of proteins involved in translation including a cluster of downregulated ribosomal proteins. Tobradex® intervention was associated with the regulation of proteins involved in fructose metabolism and glycolytic processes including fructose‐1.6‐bisphosphatase 1, fructose‐bisphosphate aldolases A and B, pyruvate kinase PKM and transketolase. Ig lambda chain V‐I region, prominin‐1, and protein Niban were upregulated after Tobradex® treatment. Conclusions: Tobradex treatment reversed the expression of plakin proteins in anterior blepharitis. Topical solutions which inhibit the expression of plakin proteins may have the potential to restore the ocular surface integrity in anterior blepharitis and should be explored further. [ABSTRACT FROM AUTHOR]

Background: The proteome of the peri‐implant crevicular fluid (PICF) has not been systematically investigated. The aim of the present study was to reveal the proteome biology of dental implants affected with peri‐implantitis. Methods: Patients with at least one diseased implant were included (probing depth ≥6 mm, ≥3 mm peri‐implant radiological bone loss). Using sterile paper strips, samples were collected from healthy implants (I), healthy teeth (T) and peri‐implantitis affected implants (P). Proteome analysis was performed using liquid chromatography — tandem mass spectrometry (LC‐MS/MS) and data independent acquisition, allowing the identification and quantification of human and bacterial proteins as well as semi‐specific peptides. Results: A total of 38 samples from 14 patients were included in the study; 2332 different human proteins were identified across all samples. No differentially expressed proteins between T and I were found. Comparing P to I, 59 proteins were found upregulated and 31 downregulated in P with significance. Upregulated proteins included proinflammatory proteins such as immunoglobulins, dysferlin, and S100P, as well as antimicrobial proteins, for example, myeloperoxidase or azurocidin. Gene ontology analysis further revealed higher activity of immunological pathways. Proteolytic patterns indicated the activity of inflammatory proteins such as cathepsin G. A total of 334 bacterial proteins were identified and quantified. Peri‐implantitis showed elevated proteolytic activity. Conclusion: I and T share similarities in their proteome, while diseased implants deviate strongly from healthy conditions. The PICF proteome of peri‐implantitis affected sites exhibits an inflammatory fingerprint, dominated by neutrophil activity when compared with healthy implants. [ABSTRACT FROM AUTHOR]

Rationale: The observed isotope distribution is an important attribute for the identification of peptides and proteins in mass spectrometry-based proteomics. Sulphur atoms have a very distinctive elemental isotope definition, and therefore, the presence of sulphur atoms has a substantial effect on the isotope distribution of biomolecules. Hence, knowledge of the number of sulphur atoms can improve the identification of peptides and proteins. Methods: In this paper, we conducted a theoretical investigation on the isotope properties of sulphur-containing peptides. We proposed a gradient boosting approach to predict the number of sulphur atoms based on the aggregated isotope distribution. We compared prediction accuracy and assessed the predictive power of the features using the mass and isotope abundance information from the first three, five and eight aggregated isotope peaks. Results: Mass features alone are not sufficient to accurately predict the number of sulphur atoms. However, we reach near-perfect prediction when we include isotope abundance features. The abundance ratios of the eighth and the seventh, the fifth and the fourth, and the third and the second aggregated isotope peaks are the most important abundance features. The mass difference between the eighth, the fifth or the third aggregated isotope peaks and the monoisotopic peak are the most predictive mass features. Conclusions: Based on the validation analysis it can be concluded that the prediction of the number of sulphur atoms based on the isotope profile fails, because the isotope ratios are not measured accurately. These results indicate that it is valuable for future instrument developments to focus more on improving spectral accuracy to measure peak intensities of higher-order isotope peaks more accurately. [ABSTRACT FROM AUTHOR]

BACKGROUND: Scrophularia ningpoensis is a well‐known medicinal crop. Continuous cropping seriously affects the yield and quality, but little is known about the influence of continuous cropping on metabolic pathways. In this study, the difference in protein abundance between continuous cropping and non‐continuous cropping of S. ningpoensis roots was studied by proteomics, and the molecular mechanism that protects S. ningpoensis against continuous cropping was explored. RESULTS: The results suggested that continuous cropping in S, ningpoensis altered the expression of proteins related to starch and sucrose metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, citric acid cycle, phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid biosynthesis, terpenoid backbone biosynthesis, monoterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, and steroid biosynthesis. Among these processes, the most affected were phenylpropanoid biosynthesis and starch and sucrose metabolism, which may be important for continuous cropping resistance. CONCLUSION: The effect of continuous cropping on S. ningpoensis was demonstrated at the proteome level in this work, and identified candidate proteins that may cause continuous cropping reactions. The paper provides the theoretical foundation and scientific reference for enhancing the continuous cropping resistance of S. ningpoensis. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Overdiagnosis of early melanoma is a significant problem. Due to subtle unique and overlapping clinical and histological criteria between pigmented lesions and the risk of mortality from melanoma, some benign pigmented lesions are diagnosed as melanoma. Although histopathology is the gold standard to diagnose melanoma, there is a demand to find alternatives that are more accurate and cost‐effective. In the current "omics" era, there is gaining interest in biomarkers to help diagnose melanoma early and to further understand the mechanisms driving tumor progression. Genomic investigations have attempted to differentiate malignant melanoma from benign pigmented lesions. However, genetic biomarkers of early melanoma diagnosis have not yet proven their value in the clinical setting. Protein biomarkers may be more promising since they directly influence tissue phenotype, a result of by‐products of genomic mutations, posttranslational modifications and environmental factors. Uncovering relevant protein biomarkers could increase confidence in their use as diagnostic signatures. Currently, proteomic investigations of melanoma progression from pigmented lesions are limited. Studies have previously characterised the melanoma proteome from cultured cell lines and clinical samples such as serum and tissue. This has been useful in understanding how melanoma progresses into metastasis and development of resistance to adjuvant therapies. Currently, most studies focus on metastatic melanoma to find potential drug therapy targets, prognostic factors and markers of resistance. This paper reviews recent advancements in the genomics and proteomic fields and reports potential avenues, which could help identify and differentiate melanoma from benign pigmented lesions and prevent the progression of melanoma. [ABSTRACT FROM AUTHOR]

Aim: An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates. Materials and results: The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7 cm IPG strips of pH 4‐7 and SDS‐PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was Western blotted to nitrocellulose paper (NCP) and reacted with rohu anti‐A siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver‐stained gel and subjected to mass spectrometry in MALDI‐TOF/TOF. The MS/MS spectra were analysed in MASCOT software with taxonomy 'other metazoa' and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in 'molecular function' and belonging to metabolic processes in 'biologic process' categories. The possibility of these proteins as vaccine candidates against Asiamensis is discussed in the paper. Conclusion: Three of the identified proteins namely, bromodomain‐containing protein, anaphase‐promoting complex subunit 5 and elongation factor‐2 could possibly serve as vaccine candidates against argulosis in carps. [ABSTRACT FROM AUTHOR]

Background: This paper compares experimental findings and simulation outcomes of single and multiple protein models moving through a nanopipette biosensor. It provides insights into the factors influencing the process and explores their relevance to proteomics. Methods: Nanopipette biosensors were produced by pulling borosilicate glass tubes and treating them with an electron beam. A scanning electron microscope was used to characterize the nanopipettes. The study measured and modeled ionic currents for the elastase‐specific inhibitor protein. Simulation models were developed using the finite element method and Poisson–Boltzmann formalism, considering different protein configurations and translocation scenarios. Results: The results showed that the pore current of a nanopipette decreases as the protein approaches the nanopipette. The minimum pore current occurs at the widest part of the protein, and the current increases as the protein progresses through the nanopipette. For multiple protein translocations, the pore current decreases between the widest parts of the first and second proteins, and the lowest current is observed at the broadest part of the second protein. After the third protein, the pore current remains constant. It is also found that the fractional blockade difference, translocation speed, fluctuation in pore current, and dwell time are all affected by the number of proteins translocating through the nanopipette. The fractional blockade difference, the decrease in pore current caused by the protein, increases with the number of proteins while the translocation speed decreases. The fluctuation in pore current and dwell time is also longer for three‐protein translocations than for single‐protein translocations. Conclusion: This study offers valuable insights into biomolecule transport through nanopipettes, enhances our understanding of protein dynamics in restricted environments, and significantly contributes to single‐protein sequencing studies, drug screening, and proteomics. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic symmetrical multiple arthritis. Current traditional counter‐therapies are expensive and have side effects. Xanthii Fructus has effects in expelling wind and cold, draining the nasal orifice, and removing wind and dampness. However, its mechanism of action against rheumatoid arthritis is unknown. In this paper, the mechanism of the anti‐ rheumatoid arthritis effect of Xanthii Fructus is studied by proteomics. The experimental results show that it could significantly reduce serum inflammatory factor levels, alleviate joint edema, improve vasodilation and congestion, and significantly reduce the number of inflammatory cells. Proteomics results show that the PI3K‐AKT signaling pathway is the key pathway for Xanthii Fructus to treat rheumatoid arthritis. In this study, we obtained a new understanding of the mechanism of Xanthii Fructus in the treatment of rheumatoid arthritis, which provided a theoretical basis for its prevention and treatment and laid the foundation for further research. [ABSTRACT FROM AUTHOR]

Background: Oral lichen planus (OLP), a World Health Organization (WHO)‐classified oral potentially malignant condition, confers a 1% risk of transformation to oral squamous cell carcinoma (OSCC). There does not appear to be a consensus understanding of the underlying molecular events. This scoping review aimed to identify critical molecular pathways and highlight gaps in existing knowledge on malignant transformation in OLP. Methods: Following Preferred Reporting Items for Systematic Reviews and Meta‐Analysis Protocols (PRISMA‐P) guidelines, a comprehensive literature search and methodical screening identified 61 relevant studies detailing molecular differences between OLP and OSCC. Results: Molecular changes shared between OLP and OSCC included those affecting cellular proliferation (altered p53 expression, hypermethylation of p16/CDKN2A, MYC gains, increased ki‐67), apoptosis (increased bcl‐2 and survivin expression), extracellular matrix (ECM) remodeling (increased matrix metalloproteinase [MMP] expression), and transcriptional control (altered bmi1 and microRNA [miRNA] expression). In addition, some molecular alterations accumulated incrementally from control to OLP to OSCC or were present in higher‐risk erosive variants of OLP or transformed OLP. Few studies included rigorous diagnostic inclusion criteria or unbiased discovery methods. Conclusions: Results of this review support the potentially malignant nature of OLP and imply that molecular events associated with malignant transformation may be heterogeneous. In addition, findings in this review highlight the need for additional studies using rigorous diagnostic inclusion criteria and unbiased discovery methods to further understand this process. [ABSTRACT FROM AUTHOR]

CHIP (C‐terminus of Hsc70‐interacting protein) and its worm ortholog CHN‐1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin‐proteasome system (UPS). CHN‐1 can cooperate with UFD‐2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN‐1–UFD‐2 complex in Caenorhabditis elegans. Our data show that UFD‐2 binding promotes the cooperation between CHN‐1 and ubiquitin‐conjugating E2 enzymes by stabilizing the CHN‐1 U‐box dimer. However, HSP70/HSP‐1 chaperone outcompetes UFD‐2 for CHN‐1 binding, thereby promoting a shift to the autoinhibited CHN‐1 state by acting on a conserved residue in its U‐box domain. The interaction with UFD‐2 enables CHN‐1 to efficiently ubiquitylate and regulate S‐adenosylhomocysteinase (AHCY‐1), a key enzyme in the S‐adenosylmethionine (SAM) regeneration cycle, which is essential for SAM‐dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN‐1 and UFD‐2 in substrate ubiquitylation. Synopsis: The regulation of CHIP quality control ubiquitin ligase activity and its substrate selectivity is largely unclear. Here, biochemical, computational, proteomic, and lipidomic data unravel the regulatory mechanism of CHIP processivity, and reveal its role in lipid metabolism. The E3 ligase UFD‐2 stimulates the ubiquitylation activity of CHIP/CHN‐1UFD‐2 binding promotes dimerization of the CHIP/CHN‐1 U‐box domains and E2 enzyme discharging capacityHSP70/HSP‐1, by latching the U‐box and TPR domains, stabilizes the autoinhibitory state of CHIP/CHN‐1, thus limiting its interactions with E2s and UFD‐2Assembly with UFD‐2 enables CHIP/CHN‐1 to regulate lipid metabolism via S‐adenosylhomocysteinase (AHCY‐1) ubiquitylation [ABSTRACT FROM AUTHOR]

Dr Mischak: "Re-analysis of peptidomic analysis of cartilage and subchondral bone in OA patients" In May 2019 issue of European Journal of Clinical Investigations, we in our paper I Peptidomic analysis of cartilage and subchondral bone in OA patients i 1 reported a pilot study which primarily is a protocol for endogenous peptide extraction from cartilage and subchondral bone from OA patients. Peptidomic analysis of cartilage and subchondral bone in OA patients. [Extracted from the article]

Parkinson's disease (PD) affects a significant proportion of the population over the age of 60 years, and its prevalence is increasing. While symptomatic treatment is available for motor symptoms of PD, non‐motor complications such as dementia result in diminished life quality for patients and are far more difficult to treat. In this study, we analyzed PD‐associated alterations in the hippocampus of PD patients, since this brain region is strongly affected by PD dementia. We focused on synapses, analyzing the proteome of post‐mortal hippocampal tissue from 16 PD cases and 14 control subjects by mass spectrometry. Whole tissue lysates and synaptosomal fractions were analyzed in parallel. Differential analysis combined with bioinformatic network analyses identified neuronal pentraxin 1 (NPTX1) to be significantly dysregulated in PD and interacting with proteins of the synaptic compartment. Modulation of NPTX1 protein levels in primary hippocampal neuron cultures validated its role in synapse morphology. Our analysis suggests that NPTX1 contributes to synaptic pathology in late‐stage PD and represents a putative target for novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

As of August 27, 2021, the ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), has spread to over 220 countries, areas, and territories. Thus far, 214,468,601 confirmed cases, including 4,470,969 deaths, have been reported to the World Health Organization. To combat the COVID-19 pandemic, multiomics-based strategies, including genomics, transcriptomics, proteomics, andmetabolomics, have been used to study the diagnosismethods, pathogenesis, prognosis, and potential drug targets of COVID-19. In order to help researchers and clinicians to keep up with the knowledge of COVID-19, we summarized the most recent progresses reported in omics-based research papers. This review discusses omics-based approaches for studying COVID-19, summarizing newly emerged SARS-CoV-2 variants as well as potential diagnostic methods, risk factors, and pathological features of COVID-19. This review can help researchers and clinicians gain insight into COVID-19 features, providing direction for future drug development and guidance for clinical treatment, so that patients can receive appropriate treatment as soon as possible to reduce the risk of disease progression. [ABSTRACT FROM AUTHOR]

Summary: Plant Ecophysiology is the study on how Plant Physiology is modulated by the environment. This discipline could have benefited greatly from the development of the different 'omic' technologies (from genomics to metabolomics). Instead, the overall impression is that ecophysiology and 'omics' have developed mostly independent each other. Here we provide a literature analysis over the past 20 years which fully confirms this view. Then, we review a few examples of studies in which ecophysiology and 'omics' studies have combined to different extents to illustrate the potential benefits from their mutualistic interaction. In addition, we debate on the possibilities of working with plants other than Arabidopsis, which is illustrated with some examples of fascinating plants from extreme environments of the world, what we call the 'sherplants'. Finally, we raise a call to both communities (ecophysiology and 'omics') to integrate these disciplines to enter an 'ecophysiolomics era' to maximize our understanding about plant mechanisms from a multidisciplinary approach. Significance Statement: In this work we analyzed the relationship between the 'omic' technologies and plant ecophysiology. Literature data show that they developed mostly independent each other over the last 20 years. This fact promotes separated communities in each field losing the potential benefits from their mutualistic interaction. Here, we raise a call to both communities to integrate these disciplines to maximize our understanding about plant mechanisms from a multidisciplinary approach. [ABSTRACT FROM AUTHOR]

Abstract: Glycomics lags substantially behind proteomics and genomics in its ability to decipher and synthesize complex glycans. The slow progress in deciphering glycan interactions at a molecular level is in large part due to the absence of a functional system to express, on a large scale, carbohydrates of known structure, in the context of a biologically relevant assay system. Here, the characterization of glycan‐functionalized catanionic surfactant vesicles (CVs) as a platform for glycan synthesis is described, and it is demonstrated that the resulting glycan‐functionalized CVs can serve as a scaffold for the interrogation of protein‐glycan interactions. It is demonstrated that Neisseria gonorrhoeae lipooligosaccharide (LOS) glycosyltransferase LgtE, an enzyme that catalyzes the addition of galactose onto a terminal glucose found on LOS, can be used to biochemically modify LOS or glucose functionalized CVs. CVs are characterized by differential lectin binding using flow cytometry. LgtE activity is measured on whole cells and LOS functionalized vesicles and found to have approximately the same biochemical properties. It is further demonstrated that CVs can be inkjet printed. This paper presents proof‐of‐concept that glycan‐functionalized catanionic vesicles can be used to create a high‐specificity and high‐throughput glycan array that will allow for the investigation of a variety of protein–glycan interactions. [ABSTRACT FROM AUTHOR]

Herein, the utility of Raman spectroscopy for rapid identity (ID) verification of biotherapeutic protein products in solution is demonstrated. The success of this approach hinges on the unique combination of Raman features associated with both the therapeutic agent and excipients as the basis for product differentiation. Product ID method development subjects Raman spectra of the target product(s) to dimension reduction using principal component analysis (PCA) to define product‐specific models which serve as the basis for the ID method decision algorithm. In the initial stages of this work, instrumental sources of variance such as intensity‐axis multiplicative scaling and wavenumber‐axis alignment lead to high rates of erroneous false‐negatives when the methods were executed on other instruments that were not used for model development. To overcome these issues, the PCA‐based spectral models were systematically studied and iteratively adjusted to elucidate key design requirements for transferable models. The use of specific spectral preprocessing algorithms was implemented to compensate for instrumental variance, which reduced false‐negative rates without sacrificing method specificity. Five different methods, along with the associated product samples, were evaluated on 15 Raman instruments that had not been used for method development. No false‐positive analyses occurred for any of the five methods. A small number of false negatives (~2%) were observed on analyzers that performed outside of the instrument manufacturer's specifications and thus would have been identified during preventive maintenance. Taken together, this paper lays the groundwork for developing and implementing Raman ID methods for solution‐based protein products in the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

Serological screening of sexual assault evidence has traditionally focused on enzyme activity and immunochromatographic assays that provide only a presumptive indication of seminal fluid and have limited sensitivity relative to DNA testing. Seminal fluid detection based on protein mass spectrometry represents a "Next Gen" serological technology that overcomes the specificity and sensitivity limitations of traditional serological screening but requires time‐consuming sample preparation protocols. This paper describes a novel "peptidomics" approach to seminal fluid detection that eliminates the need for lengthy trypsin digestion. This streamlines sample preparation to a one‐step process followed by high‐resolution mass spectrometry to identify naturally occurring seminal fluid peptides and low‐molecular weight proteins. Multiple protein biomarkers of seminal fluid were consistently and confidently identified based on the multiplexed detection of numerous endogenous peptides. These included Semenogelin I and II (90% and 86% sequence coverage, respectively); Prostate Specific Antigen/p30 (29% sequence coverage); and Prostatic Acid Phosphatase (24% sequence coverage). The performance of this streamlined peptidomics approach to seminal fluid identification in a forensic context was also assessed using simulated casework samples of the type typically collected as part of a sexual assault examination (e.g., oral and vaginal swabs stained with semen). The resulting data demonstrate that sub‐microliter quantities of seminal fluid on cotton swabs can be recovered and reliably detected. This supports the forensic applicability of a peptidomic assay for seminal fluid identification with same‐day sample preparation and analysis. Future development and streamlined multiplex peptidomic assays for additional biological stains can easily be envisaged. [ABSTRACT FROM AUTHOR]

Background The exact causes of polycystic ovary syndrome (PCOS) are uncertain, and treatment could be improved. Discovery-based approaches like ‘proteomics’ may result in faster insights into the causes of PCOS and improved treatment. Objectives To identify the number and nature of proteomic biomarkers found in PCOS so far and to identify their diagnostic and therapeutic potential. Search strategy All published studies on proteomic biomarkers in women with PCOS identified through the MEDLINE (1966–2008), EMBASE (1980–2008) and the ISI web of knowledge (v4.2) databases. Selection criteria The terms ‘polycystic ovary syndrome’ and ‘proteomic’, ‘proteomics’, ‘proteomic biomarker’ or ‘proteomics biomarker’ without any limits/restrictions were used. Data collection and analysis Original data were abstracted where available and summarised on a separate Microsoft Excel (2007) database for analysis. Main results Seventeen articles were identified, of which 6 original papers and 1 review article contained original data. Tissues investigated included serum, omental biopsies, ovarian biopsies, follicular fluid and T lymphocytes. Sample sizes ranged from 3 to 30 women. One hundred and forty-eight biomarkers were identified. The biomarkers were involved in many pathways, for example the regulation of fibrinolysis and thrombosis, insulin resistance, immunity/inflammation and the antioxidant pathway. Eleven groups of biomarkers appeared to be independently validated. The individual sensitivities for the diagnosis of PCOS were reported for 11 named biomarkers and ranged from 57 to 100%. Author’s conclusions Proteomic biomarker discovery in PCOS offers great potential. Current challenges include reproducibility and data analysis. The establishment of a PCOS-specific biomarker data bank and international consensus on the framework of systematic reviews in this field are required. [ABSTRACT FROM AUTHOR]

Mediator‐based extracellular electron transfer (EET) pathways can balance the redox metabolism of microbes. However, such electro‐biosynthesis processes are constrained by the unknown underlying EET mechanisms. In this paper, Pseudomonas putida was studied to systematically investigate its EET pathway to transition metal complexes (i. e., [Fe(CN)6]3−/4− and [Co(bpy)3]3+/2+; bpy=2,2′‐bipyridyl) under anaerobic conditions. Comparative proteomics showed the aerobic respiratory components were upregulated in a bioelectrochemical system without oxygen, suggesting their potential contribution to EET. Further tests found inhibiting cytochrome c oxidase activity by NaN3 and NADH dehydrogenase by rotenone did not significantly change the current output. However, the EET pathway was completely blocked, while cytochrome c reductase activity was inhibited by antimycin A. Although it cannot be excluded that cytochrome c and the periplasmic subunit of cytochrome c oxidase donate electrons to the transition metal complexes, these results strongly demonstrate that cytochrome c reductase is a key complex for the EET pathway. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus is the most common type of diabetes, and insulin resistance (IR) is its core pathological mechanism. Proteomics is an ingenious and promising Omics technology that can comprehensively describe the global protein expression profiling of body or specific tissue, and is widely applied to the study of molecular mechanisms of diseases. In this paper, we focused on insulin target organs: adipose tissue, liver, and skeletal muscle, and analyzed the different pathological processes of IR in these three tissues based on proteomics research. By literature studies, we proposed that the main pathological processes of IR among target organs were diverse, which showed unique characteristics and focuses. We further summarized the differential proteins in target organs which were verified to be related to IR, and discussed the proteins that may play key roles in the emphasized pathological processes, aiming at discovering potentially specific differential proteins of IR, and providing new ideas for pathological mechanism research of IR. [ABSTRACT FROM AUTHOR]

Introduction: Dental pulp with special structure has become a good reference sample in paleomicrobiology‐related blood‐borne diseases, many pathogens were detected by different methods based on the diagnosis of nucleic acids and proteins. Objectives: This review aims to propose the preparation process from ancient teeth collection to organic molecule extraction of dental pulp and summary, analyze the methods that have been applied to detect septicemic pathogens through ancient dental pulps during the past 20 years following the first detection of an ancient microbe. Methods: The papers used in this review with two main objectives were obtained from PubMed and Google scholar with combining keywords: "ancient," "dental pulp," "teeth," "anatomy," "structure," "collection," "preservation," "selection," "photography," "radiography," "contamination," "decontamination," "DNA," "protein," "extraction," "bone," "paleomicrobiology," "bacteria," "virus," "pathogen," "molecular biology," "proteomics," "PCR," "MALDI‐TOF," "LC/MS," "ELISA," "immunology," "immunochromatography," "genome," "microbiome," "metagenomics." Results: The analysis of ancient dental pulp should have a careful preparation process with many different steps to give highly accurate results, each step complies with the rules in archaeology and paleomicrobiology. After the collection of organic molecules from dental pulp, they were investigated for pathogen identification based on the analysis of DNA and protein. Actually, DNA approach takes a principal role in diagnosis while the protein approach is more and more used. A total of seven techniques was used and ten bacteria (Yersinia pestis, Bartonella quintana, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi C, Mycobacterium leprae, Mycobacterium tuberculosis, Rickettsia prowazeki, Staphylococcus aureus, Borrelia recurrentis, Bartonella henselae) and one virus (Anelloviridae) were identified. Y. pestis had the most published in quantity and all methods were investigated for this pathogen, S. aureus and B. recurrentis were identified by three different methods and others only by one. The combining methods interestingly increase the positive rate with ELISA, PCR and iPCR in Yersinia pestis diagnosis. Twenty‐seven ancient genomes of Y. pestis and one ancient genome of B. recurrentis were reconstructed. Comparing to the ancient bone, ancient teeth showed more advantage in septicemic diagnosis. Beside pathogen identification, ancient pulp help to distinguish species. Conclusions: Dental pulp with specific tissue is a suitable sample for detection of the blood infection in the past through DNA and protein identification with the correct preparation process, furthermore, it helps to more understand the pathogens of historic diseases and epidemics. [ABSTRACT FROM AUTHOR]

Mass mortality events are routinely reported in bivalve cultivation practices worldwide with significant economic consequences for the global aquaculture industry. Major contributors of these bivalve losses are infections caused by viruses and bacteria whose molecular action mechanisms are still not fully understood. This major bottleneck for shellfish cultivation presents an opportunity for fundamental research that can generate practical tools for reliable health assessments. To this end, newly developed omics approaches are likely to facilitate knowledge acquisition, especially if applied in conjunction with established and emergent immunological techniques. In recent years, various omics technologies (e.g. genomics, transcriptomics, proteomics and metabolomics) have been used to study the Pacific oyster mortality events associated with detection of ostreid herpesvirus type 1 (OsHV‐1) and Vibrio bacteria. These new approaches, especially when used in combination have significantly improved our understanding of this syndrome and associated diseases. In this paper, we review the current omics technologies that have been employed to investigate the interactions between Pacific oyster Crassostrea gigas and pathogens (e.g. OsHV‐1 and Vibrio sp.), infection mechanisms, host susceptibility and resistance. Herein, we also highlight some of the current challenges and future directions of omics approaches in shellfish health research, including disease detection, diagnosis and treatment. [ABSTRACT FROM AUTHOR]

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research and perspectives on the journal and more. Albert Heck is Professor of Chemistry and Pharmaceutical Sciences at Utrecht University, Scientific Director of the Netherlands Proteomics Center, and Head of the Biomolecular Mass Spectrometry and Proteomics group in Utrecht University since September 1998. He has served as Editorial Board Member of The FEBS Journal since 2020. [ABSTRACT FROM AUTHOR]

Many cellular processes are governed by protein–protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein–protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes. Synopsis: A new temporal proteomics technique reveals the protein interaction dynamics of folding and secretion pathways. The method clarifies how mutant forms of the pro-hormone thyroglobulin result in congenital hypothyroidism by elucidating the defects in protein quality control. TRIP combines pulsed unnatural amino acid labeling, biotin-streptavidin enrichment, and TMTpro-multiplexed proteomics to study time-resolved protein interactions. Secretion-defective thyroglobulin mutants exhibit prolonged interaction dynamics with chaperone, disulfide exchange, and degradation pathway interactions. Depletion of degradation factors VCP and TEX264 rescues mutant prohormone secretion. VCP inhibition shifts temporal protein interactions to rescue mutant thyroglobulin secretion. A new temporal proteomics technique reveals the protein interaction dynamics of folding and secretion pathways. The method clarifies how mutant forms of the pro-hormone thyroglobulin result in congenital hypothyroidism by elucidating the defects in protein quality control. [ABSTRACT FROM AUTHOR]

Crosslinking mass spectrometry is a powerful tool to study protein-protein interactions under native or near-native conditions in complex mixtures. Through novel search controls, we show how biassing results towards likely correct proteins can subtly undermine error estimation of crosslinks, with significant consequences. Without adjustments to address this issue, we have misidentified an average of 260 interspecies protein-protein interactions across 16 analyses in which we synthetically mixed data of different species, misleadingly suggesting profound biological connections that do not exist. We also demonstrate how data analysis procedures can be tested and refined to restore the integrity of the decoy-false positive relationship, a crucial element for reliably identifying protein-protein interactions. Synopsis: Accurate error estimation in crosslinking mass spectrometry (MS) is crucial for uncovering new biology. However, new data analysis methods may disrupt the decoy-false positive relationship that is at the basis of error estimation. This study offers tests and remedies to address this issue. Crosslinking MS error control easily breaks, obscuring true errors and inflating novel biology claims. Concatenated data and true positive estimation reveal data processing errors. Pairing targets and decoys throughout the analysis improves the robustness of error estimation. Accurate error estimation in crosslinking mass spectrometry (MS) is crucial for uncovering new biology. However, new data analysis methods may disrupt the decoy-false positive relationship that is at the basis of error estimation. This study offers tests and remedies to address this issue. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer often associated with chronic hepatitis B virus infection (CHB) and liver cirrhosis (LC), underscoring the critical need for biomarker discovery to improve patient outcomes. Emerging as a promising avenue for biomarker development, proteomic technology leveraging liquid biopsy from small extracellular vesicles (sEV) offers new insights. Here, we evaluated various methods for sEV isolation and identified polysaccharide chitosan (CS) as an optimal approach. Subsequently, we employed optimized CS‐based magnetic beads (Mag‐CS) for sEV separation from serum samples of healthy controls, CHB, LC, and HBV‐HCC patients. Leveraging data‐independent acquisition mass spectrometry coupled with machine learning, we uncovered potential vesicular protein biomarker signatures (KNG1, F11, KLKB1, CAPNS1, CDH1, CPN2, NME2) capable of distinguishing HBV‐HCC from CHB, LC, and non‐HCC conditions. Collectively, our findings highlight the utility of Mag‐CS‐based sEV isolation for identifying early detection biomarkers in HBV‐HCC. [ABSTRACT FROM AUTHOR]

Introduction: Modic changes (MC) are bone marrow lesions of vertebral bones, which can be detected with magnetic resonance imaging (MRI) adjacent to degenerated intervertebral discs. Defined by their appearance on T1 and T2 weighted images, there are three interconvertible types: MC1, MC2, and MC3. The inter‐observer variability of the MRI diagnosis is high, therefore a diagnostic serum biomarker complementing the MRI to facilitate diagnosis and follow‐up would be of great value. Methods: We used a highly sensitive and reproducible proteomics approach: DIA/SWATH‐MS to find serum biomarkers in a subset of the Northern Finland Birth Cohort 1966. Separately, we measured a panel of factors involved in inflammation and angiogenesis to confirm some potential biomarkers published before with an ELISA‐based method called V‐Plex. Results: We found neither an association between the serum concentrations of the proteins detected with DIA/SWATH‐MS with the presence of MC, nor a correlation with the size of the MC lesions. We did not find any association between the factors measured with the V‐Plex and the presence of MC or their size. Conclusion: Altogether, our study suggests that a robust and generally usable biomarker to facilitate the diagnosis of MC cannot readily be found in serum. [ABSTRACT FROM AUTHOR]

Cellular signaling regulates various cellular functions via protein phosphorylation. Phosphoproteomic data potentially include information for a global regulatory network from signaling to cellular functions, but a procedure to reconstruct this network using such data has yet to be established. In this paper, we provide a procedure to reconstruct a global regulatory network from signaling to cellular functions from phosphoproteomic data by integrating prior knowledge of cellular functions and inference of the kinase–substrate relationships (KSRs). We used phosphoproteomic data from insulin‐stimulated Fao hepatoma cells and identified protein phosphorylation regulated by insulin specifically over‐represented in cellular functions in the KEGG database. We inferred kinases for protein phosphorylation by KSRs, and connected the kinases in the insulin signaling layer to the phosphorylated proteins in the cellular functions, revealing that the insulin signal is selectively transmitted via the Pi3k‐Akt and Erk signaling pathways to cellular adhesions and RNA maturation, respectively. Thus, we provide a method to reconstruct global regulatory network from signaling to cellular functions based on phosphoproteomic data. In this study, we reconstruct global regulatory network from insulin signaling to the cellular functions via protein phosphorylation based on phosphoproteomic data. The arrows indicate phosphorylation of the quantitatively changed phosphopeptides by the protein kinases. [ABSTRACT FROM AUTHOR]

Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error‐controlled, complex‐centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub‐complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity. Synopsis: The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control. The presented workflow is based on the concept of complex‐centric proteome profiling and combines size exclusion chromatography (SEC) with SWATH/DIA mass spectrometry.The implementation of the complex‐centric data analysis is supported by the computational framework CCprofiler.Application of the SEC‐SWATH‐MS workflow to HEK293 cells led the detection and quantification of subunit distribution of 462 distinct protein complexes containing 2,127 proteins and identified novel complex variants such as assembly intermediates.The interactive platform, SECexplorer is presented to support custom complex‐centric exploration of SEC‐SWATH‐MS datasets. The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control. [ABSTRACT FROM AUTHOR]

A cationic AuI N‐heterocyclic carbene complex featuring caffeine‐based ligands has shown potent and selective anticancer effects in ovarian cancer cells. This organogold compound is remarkably stable in physiological conditions. Therefore, the observed therapeutic effects are mainly due to non‐covalent interactions of the gold compound with its pharmacological targets. Among the latter, DNA secondary structures—G‐quadruplexes—play a pivotal role in the mechanisms of anticancer action. More information can be found in the Full Paper by S. M. Meier‐Menches, A. Casini, C. Gerner, et al. on page 15528. [ABSTRACT FROM AUTHOR]

Abstract: It is quite a challenge to develop model‐free feature screening approaches for missing response problems because the existing standard missing data analysis methods cannot be applied directly to high dimensional case. This paper develops some novel methods by borrowing information of missingness indicators such that any feature screening procedures for ultrahigh‐dimensional covariates with full data can be applied to missing response case. The first method is the so‐called missing indicator imputation screening, which is developed by proving that the set of the active predictors of interest for the response is a subset of the active predictors for the product of the response and missingness indicator under some mild conditions. As an alternative, another method called Venn diagram‐based approach is also developed. The sure screening property is proven for both methods. It is shown that the complete case analysis can also keep the sure screening property of any feature screening approach with sure screening property. [ABSTRACT FROM AUTHOR]

Summary: Target‐Decoy database is currently the method of choice to assess the quality of Proteins' search engines. Decoy versions of real peptides are generated and injected to the same database of real ones with different labels. Quality of search engines results is assessed based on the number of decoys retrieved as hits. In Crux‐Tide search engine, which is one of the fastest search engines currently available, the process of indexing and generating decoys is computationally expensive. In this paper, we analyze the serial algorithm in detail and show improvement possibilities, and then describe a parallel‐shared memory solution using OpenMP. To completely break up the dependency in the serial algorithms, a clever hashing technique is utilized to localize the process. The parallel solution and the hashing technique together are able to reduce the computation cost by approximately 70‐80% using few threads. Besides the parallelization, we redesign part of the serial code so that the memory consumption becomes more efficient. The parallel version can index the same files using around two‐third of the memory space that the serial version consumes. This solution could impact and support future distributed developments of Crux‐Tide searching phase, where each parallel unit could rank the observed spectra independently. [ABSTRACT FROM AUTHOR]

Vityaz at the Crossroads by Viktor Mikhailovich Vasnetsov, 1882. Decision making is an important part of our lives, and chemometrics contributes to making choices more purposeful. Sometimes choosing the wrong way can lead to serious trouble. As written on the stone in the illustration, “If you ride to the left, you will lose your horse, if you ride to the right, you will lose your head.” Partial least squares discriminant analysis (PLS‐DA) is an enormously popular method in various scientific areas: genomics, proteomics, metabolomics, and in food and pharmaceutical sciences. In this paper, we provide a detailed PLS‐DA theory that could be used as an itinerary in this perilous world. A distinct feature of this theory is that it does not utilize the PLS scores but is entirely based on the predicted dummy responses. We demonstrate that the results of the direct multi‐class PLS‐DA can be presented in a straightforward way by projecting the response matrix on the “super‐score” space by means of Principal Component Analysis. Two approaches to discrimination are considered: the hard and the soft way of allocation. Correspondingly, two versions of PLS‐DA are presented: the conventional hard PLS‐DA and the newly introduced soft PLS‐DA that seems to be a novel approach in chemometrics. The quality of classification is assessed using figures of merit (sensitivity, specificity, and efficiency). It is shown how these characteristics are used for the selection of the model complexity. A number of practical problems are scrutinized, such as unbalanced sizes of classes, comparison of discriminant and class‐modeling approaches, and authentication by the “one against all” strategy. The paper is illustrated by real‐world and simulated examples. [ABSTRACT FROM AUTHOR]

Understanding how porous biomaterials interact with cells at their surface and how they either promote or inhibit cellular processes has presented several challenges. Additive manufacturing enables the fabrication of scaffolds with distinct compositions and designs for different tissue engineering applications. To evaluate the in vitro performance of multiple printed materials, biochemical assays can be limiting in providing valuable insight and key information to select the best tissue destination. Omics technologies like proteomics are crucial for studying important cellular events and gathering valuable information about cellular processes and mechanisms. However, only few studies focus on proteomics to decipher cell–material interactions and cell differentiation on additive manufactured scaffolds. Here, scaffolds were fabricated using three polymers (polycaprolactone (PCL), poly(ethylene oxide)–poly(butylene terephthalate) (PEOT/PBT), and polylactic acid (PLA)) through additive manufacturing. Their chondrogenic and osteogenic potential were characterized and compared using human bone marrow‐derived mesenchymal stem cells (hBMSCs) through proteomics analysis. The 3D scaffolds were all hydrophilic and displayed Young's moduli close to those of bone or cartilage for PLA and PCL and PEOT/PBT, respectively. Biochemical assays indicated that PEOT/PBT and PLA scaffolds have a greater chondrogenic potential by higher glycosaminoglycan (GAG) and collagen deposition compared to PCL. PLA and PEOT/PBT showed to be more effective in promoting bone formation, as evidenced by higher calcium deposits detected by alizarin red staining, and higher alkaline phosphatase (ALP), especially for PLA in osteogenic medium. Proteomics data revealed the most distinct separation between conditions in chondrogenic medium, which had the highest protein identification rates. Pathway analysis showed that PCL did not induce any differentiation‐related pathways when compared to PEOT/PBT and PLA in any of the tested media conditions. Analysis of PEOT/PBT proteins showed pathways involved in chondrogenesis in all three media and pathways related to hypertrophic phenotype progression in chondrogenic medium. These data suggests that PEOT/PBT is a valuable candidate for cartilage and osteochondral applications, able to drive hBMSCs differentiation without the need of growth factors. PLA was also a valuable candidate for cartilage and bone applications by upregulating both chondrogenic and osteogenic‐related proteins in maintenance and chondrogenic media. In osteogenic and maintenance media, the upregulation of angiogenic proteins makes PLA a better candidate for bone application where vascularization is key. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are emerging as promising carriers for the delivery of therapeutic biologics. Genetic engineering represents a robust strategy for loading proteins of interest into EVs. Identification of EV‐enriched proteins facilitates protein cargo loading efficiency. Many EV‐enriched proteins are sorted into EVs via an endosomal sorting complex required for transport (ESCRT)‐dependent pathway. In parallel, viruses hijack this EV biosynthesis machinery via conserved late domain motifs to promote egress from host cells. Inspired by the similarity of biogenesis between EVs and viruses, we developed a synthetic, Late domain‐based EV scaffold protein that enables the display of a set of single chain variable fragments (scFvs) on the EV surface. We named this scaffold the Late domain‐based exosomal antibody surface display platform (LEAP). We applied the LEAP scaffold to reprogramme HEK293T cell‐derived EVs to elicit T‐cell anti‐tumor immunity by simultaneously displaying αPD‐L1 and αCD3 scFvs on the EV surface (denoted as αPD‐L1×αCD3 bispecific T‐cell engaging exosomes, BiTExos). We demonstrated that αPD‐L1×αCD3 BiTExos actively redirected T cells to bind to PD‐L1+ tumor cells, promoting T‐cell activation, proliferation and tumoricidal cytokine production. Furthermore, the αPD‐L1×αCD3 BiTExos promoted T‐cell infiltration into the tumor microenvironment to mitigate the tumor burden in vivo. Our study suggested that the LEAP scaffold may serve as a platform for EV surface display and could be applied for a broad range of EV‐based biomedical applications. [ABSTRACT FROM AUTHOR]

Evechinus chloroticus (commonly known as kina) is a sea urchin species endemic to New Zealand. Its roe is a culinary delicacy to the indigenous Māori and a globally exported food product. Echinochrome A (Ech A) is a bioactive compound isolated from the waste product of kina shells and spines; however, the molecular mechanisms of Ech A bioactivity are not well understood, partly due to Ech A never being studied using unbiased genome‐wide analysis. To explore the high‐value pharmaceutical potential of kina food waste, we obtained unbiased functional genomic and proteomic profiles of yeast cells treated with Echinochrome A. Abundance was measured for 4100 proteins every 30 min for four hours using fluorescent microscopy, resulting in the identification of 92 proteins with significant alterations in protein abundance caused by Ech A treatment that were over‐represented with specific changes in DNA replication, repair and RNA binding after 30 min, followed by specific changes in the metabolism of metal ions (specifically iron and copper) from 60–240 min. Further analysis indicated that Ech A chelated iron, and that iron supplementation negated the growth inhibition caused by Ech A. Via a growth‐based genome‐wide analysis of 4800 gene deletion strains, 20 gene deletion strains were sensitive to Ech A in an iron‐dependent manner. These genes were over‐represented in the cellular response to oxidative stress, suggesting that Ech A suppressed growth inhibition caused by oxidative stress. Unexpectedly, genes integral to cardiolipin and inositol phosphate biosynthesis were required for Ech A bioactivity. Overall, these results identify genes, proteins, and cellular processes mediating the bioactivity of Ech A. Moreover, we demonstrate unbiased genomic and proteomic methodology that will be useful for characterizing bioactive compounds in food and food waste. [ABSTRACT FROM AUTHOR]

Neuroinflammation is initiated through microglial activation and cytokine release which can be induced through lipopolysaccharide treatment (LPS) leading to a transcriptional cascade culminating in the differential expression of target proteins. These differentially expressed proteins can then be packaged into extracellular vesicles (EVs), a form of cellular communication, further propagating the neuroinflammatory response over long distances. Despite this, the EV proteome in the brain, following LPS treatment, has not been investigated. Brain tissue and brain derived EVs (BDEVs) isolated from the cortex of LPS‐treated mice underwent thorough characterisation to meet the minimal information for studies of extracellular vesicles guidelines before undergoing mass spectrometry analysis to identify the differentially expressed proteins. Fourteen differentially expressed proteins were identified in the LPS brain tissue samples compared to the controls and 57 were identified in the BDEVs isolated from the LPS treated mice compared to the controls. This included proteins associated with the initiation of the inflammatory response, epigenetic regulation, and metabolism. These results allude to a potential link between small EV cargo and early inflammatory signalling. [ABSTRACT FROM AUTHOR]

Objectives: Non‐small‐cell lung carcinoma (NSCLC) is the most prevalent and lethal form of lung cancer. The need for biomarker‐informed stratification of targeted therapies has underpinned the need to uncover the underlying properties of the tumor microenvironment (TME) through high‐plex quantitative assays. Methods: In this study, we profiled resected NSCLC tissues from 102 patients by targeted spatial proteomics of 78 proteins across tumor, immune activation, immune cell typing, immune‐oncology, drug targets, cell death and PI3K/AKT modules to identify the tumor and stromal signatures associated with overall survival (OS). Results: Survival analysis revealed that stromal CD56 (HR = 0.384, P = 0.06) and tumoral TIM3 (HR = 0.703, P = 0.05) were associated with better survival in univariate Cox models. In contrast, after adjusting for stage, BCLXL (HR = 2.093, P = 0.02) and cleaved caspase 9 (HR = 1.575, P = 0.1) negatively influenced survival. Delta testing indicated the protective effect of TIM‐3 (HR = 0.614, P = 0.04) on OS. In multivariate analysis, CD56 (HR = 0.172, P = 0.001) was associated with better survival in the stroma, while B7.H3 (HR = 1.72, P = 0.008) was linked to poorer survival in the tumor. Conclusions: Deciphering the TME using high‐plex spatially resolved methods is giving us new insights into compartmentalised tumor and stromal protein signatures associated with clinical endpoints in NSCLC. [ABSTRACT FROM AUTHOR]

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+ , Nfix+/Del2 , Nfix+/Del24, NfixDel24/Del24 , Nfix+/Del140 , and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P  <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%–100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS. [ABSTRACT FROM AUTHOR]