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In recent years, advancements in deep learning techniques have significantly expanded the structural coverage of the human proteome. GalaxySagittarius-AF translates these achievements in structure prediction into target prediction for druglike compounds by incorporating predicted structures. This web server searches the database of human protein structures using both similarity- and structure-based approaches, suggesting potential targets for a given druglike compound. In comparison to its predecessor, GalaxySagittarius, GalaxySagittarius-AF utilizes an enlarged structure database, incorporating curated AlphaFold model structures alongside their binding sites and ligands, predicted using an updated version of GalaxySite. GalaxySagittarius-AF covers a large human protein space compared to many other available computational target screening methods. The structure-based prediction method enhances the use of expanded structural information, differentiating it from other target prediction servers that rely on ligand-based methods. Additionally, the web server has undergone enhancements, operating two to three times faster than its predecessor. The updated report page provides comprehensive information on the sequence and structure of the predicted protein targets. GalaxySagittarius-AF is accessible at https://galaxy.seoklab.org/sagittarius_af without the need for registration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Protein misfolding and aggregation can lead to various diseases. Recent studies have shed light on the aggregated protein in breast cancer pathology, which suggests that it is crucial to design chemical sensors that visualize protein aggregates in breast cancer, especially in clinical patient-derived samples. However, most reported sensors are constrained in cultured cell lines., Results: In this work, we present the development of two isophorone-based crystallization-induced-emission fluorophores for detecting proteome aggregation in breast cancer cell line and tissues biopsied from diseased patients, designated as A1 and A2. These probes exhibited viscosity sensitivity and recovered their fluorescence strongly at crystalline state. Moreover, A1 and A2 exhibit selective binding capacity and strong fluorescence for various aggregated proteins. Utilizing these probes, we detect protein aggregation in stressed breast cancer cells, xenograft mouse model of human breast cancer and clinical patient-derived samples. Notably, the fluorescence intensity of both probes light up in tumor tissues., Significance: The synthesized isophorone-based crystallization-induced-emission fluorophores, A1 and A2, enable sensitive detection of protein aggregation in breast cancer cells and tissues. In the future, aggregated proteins are expected to become indicators for early diagnosis and clinical disease monitoring of breast cancer., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Glycoproteome analysis at a site-specific level and proteome scale stands out as a highly promising approach for gaining insights into the intricate roles of glycans in biological systems. Recent years have witnessed an upsurge in the development of innovative methodologies tailored for precisely this purpose. Breakthroughs in mass spectrometry-based glycoproteomic techniques, enabling the identification, quantification, and systematic exploration of site-specific glycans, have significantly enhanced our capacity to comprehensively and thoroughly characterize glycoproteins. In this short review, we delve into novel tools in advancing site-specific glycoproteomic analysis and summarize pertinent studies published in the past two years. Lastly, we discuss the ongoing challenges and outline future prospects in the field, considering both the analytical strategies of mass spectrometry and the tools employed for data interpretation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Weiqian Cao reports financial support and article publishing charges were provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant. Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications. A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant's Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis. Our work with Tribolium castaneum larvae demonstrates that sometimes - depending on matrix and research question - more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

One-third of protein domains in the CATH database contain a recently discovered tertiary topological motif: non-covalent lasso entanglements, in which a segment of the protein backbone forms a loop closed by non-covalent interactions between residues and is threaded one or more times by the N- or C-terminal backbone segment. Unknown is how frequently this structural motif appears across the proteomes of organisms. And the correlation of these motifs with various classes of protein function and biological processes have not been quantified. Here, using a combination of protein crystal structures, AlphaFold2 predictions, and Gene Ontology terms we show that in E. coli, S. cerevisiae and H. sapiens that 71%, 52% and 49% of globular proteins contain one-or-more non-covalent lasso entanglements in their native fold, and that some of these are highly complex with multiple threading events. Further, proteins containing these tertiary motifs are consistently enriched in certain functions and biological processes across these organisms and depleted in others, strongly indicating an influence of evolutionary selection pressures acting positively and negatively on the distribution of these motifs. Together, these results demonstrate that non-covalent lasso entanglements are widespread and indicate they may be extensively utilized for protein function and subcellular processes, thus impacting phenotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Current sequence-based predictors of protein-binding residues (PBRs) belong to two distinct categories: structure-trained vs. intrinsic disorder-trained. Since disordered PBRs differ from structured PBRs in several ways, including ability to bind multiple partners by folding into different conformations and enrichment in different amino acids, the structure-trained and disorder-trained predictors were shown to provide inaccurate results for the other annotation type. A simple consensus-based solution that combines structure- and disorder-trained methods provides limited levels of predictive performance and generates relatively many cross-predictions, where residues that interact with other ligand types are predicted as PBRs. We address this unsolved problem by designing a novel and fast deep-learner, DeepPRObind, that relies on carefully designed modular convolutional architecture and uses innovative aggregate input features. Comparative empirical tests on a low-similarity test dataset reveal that DeepPRObind generates accurate predictions of structured and disordered PBRs and low amounts of cross-predictions, outperforming a comprehensive collection of 12 predictors of PBRs. Given the relatively low runtime of DeepPRObind (40 seconds per protein), we further validate its results based on an analysis of putative PBRs in the yeast proteome, confirming that interactions in disordered regions are enriched among hub proteins. We release DeepPRObind as a convenient web server at https://www.csuligroup.com/DeepPRObind/., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Peptide loss due to surface absorption can happen at any step in a protein analysis workflow and is sometimes especially deleterious for hydrophobic peptides. In this study, we found the LC-MS compatible surfactant, n-Dodecyl-β-D-maltoside (DDM), can maximize hydrophobic peptide recovery in various samples including single cell digests, mAb clinical PK samples, and mAb peptide mapping samples. In HeLa single cell proteomics analysis, more than half of all unique peptides identified were found only in DDM prepared samples, most of which had significantly higher hydrophobicities compared to peptides in control samples. In clinical PK studies, DDM enhanced hydrophobic complementarity-determining region (CDR) peptide signals significantly. The fold change of CDR peptides' intensity enhancement in DDM added samples compared to controls correlate with peptide retention time and hydrophobicity, providing guidance for surrogate peptide selection and peptide standard handling in PK studies. For peptide mapping analysis of mAbs, DDM can improve hydrophobic peptide signal and solution stability over 48 h in an autosampler at 4 °C, which can aid method qualification and transfer during drug development. Lastly, maximizing hydrophobic peptide recovery from samples dried in vacuo was achieved by DDM reconstitution, which provided higher signal for later eluting peaks and higher proteome coverage overall., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intrinsically disordered regions (IDRs) of proteins are often characterized by a high fraction of charged residues, but differ in their overall net charge and in the organization of the charged residues. The function-encoding information stored via IDR charge composition and organization remains elusive. Here, we aim to decipher the sequence-function relationship in IDRs by presenting a comprehensive bioinformatic analysis of the charge properties of IDRs in the human, mouse, and yeast proteomes. About 50% of the proteins comprise at least a single IDR, which is either positively or negatively charged. Highly negatively charged IDRs are longer and possess greater net charge per residue compared with highly positively charged IDRs. A striking difference between positively and negatively charged IDRs is the characteristics of the repeated units, specifically, of consecutive Lys or Arg residues (K/R repeats) and Asp or Glu (D/E repeats) residues. D/E repeats are found to be about five times longer than K/R repeats, with the longest found containing 49 residues. Long stretches of consecutive D and E are found to be more prevalent in nucleic acid-related proteins. They are less common in prokaryotes, and in eukaryotes their abundance increases with genome size. The functional role of D/E repeats and the profound differences between them and K/R repeats are discussed., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Proteome-wide identification of protein-protein interactions is a formidable task which has yet to be sufficiently addressed by experimental methodologies. Many computational methods have been developed to predict proteome-wide interaction networks, but few leverage both the sensitivity of structural information and the wide availability of sequence data. We present PEPPI, a pipeline which integrates structural similarity, sequence similarity, functional association data, and machine learning-based classification through a naïve Bayesian classifier model to accurately predict protein-protein interactions at a proteomic scale. Through benchmarking against a set of 798 ground truth interactions and an equal number of non-interactions, we have found that PEPPI attains 4.5% higher AUROC than the best of other state-of-the-art methods. As a proteomic-scale application, PEPPI was applied to model the interactions which occur between SARS-CoV-2 and human host cells during coronavirus infection, where 403 high-confidence interactions were identified with predictions covering 73% of a gold standard dataset from PSICQUIC and demonstrating significant complementarity with the most recent high-throughput experiments. PEPPI is available both as a webserver and in a standalone version and should be a powerful and generally applicable tool for computational screening of protein-protein interactions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Various snake species and snake predators have natural neutralization against snake toxins, which their antidotal abilities are commonly attributed to the intrinsic inhibitors produced by the liver, e.g., phospholipase A 2 inhibitor (PLI) and metalloproteinase inhibitor (SVMPI). Sinonatrix annularis was found to possess broad-spectrum neutralization to different snake venoms in our lab. Although the anti-venom compound PLIγ has been previously characterized in our laboratory, the mechanism of resistance of S. annularis to snake venoms remains obscure. In this research, a venom affinity chromatography was constructed by immobilizing D. acutus venom to NHS-agarose beads and applied for antitoxins mining from S. annularis. The binding capacity of the venom column was validated using a self-prepared rabbit antivenom against D. acutus. Serum and liver homogenate of S. annularis were then applied to the column, the bound components were profiled using SDS-PAGE and mass spectrometry. PLIs, snake venom metalloproteins inhibitor (SVMPI), small serum protein (SSP), heat shock proteins, etc were identified. To identify their toxin targets in D. acutus venom, a reverse separation was conducted by coupling the fractionated S. annularis serum proteins to NHS-agarose beads. Fifteen toxins of five families were captured and identified as follows: PLA 2 s, metalloproteinases, cysteine-rich secretory proteins, snake venom serine proteinases, and C-type lectins. These discoveries increased our understanding of the capacity and mechanism of the natural neutralization of S. annularis to snake venom. These natural inhibitors are medically significant due to their powerful and broad antidotal activities, which may provide alternative and promising drug candidates for snakebite treatment., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Though proteases were long regarded as nonspecific degradative enzymes, over time, it was recognized that they also hydrolyze peptide bonds very specifically with a limited substrate pool. This irreversible posttranslational modification modulates the fate and activity of many proteins, making proteolytic processing a master switch in the regulation of e.g., the immune system, apoptosis and cancer progression. N- and C-terminomics, the identification of protein termini, has become indispensable in elucidating protease substrates and therefore protease function. Further, terminomics has the potential to identify yet unknown proteoforms, e.g. formed by alternative splicing or the recently discovered alternative ORFs. Different strategies and workflows have been developed that achieve higher sensitivity, a greater depth of coverage or higher throughput. In this review, we summarize recent developments in both N- and C-terminomics and include the potential of top-down proteomics which inherently delivers information on both ends of analytes in a single analysis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mollusc shells represent excellent systems for the preservation and retrieval of genuine biomolecules from archaeological or palaeontological samples. As a consequence, the post-mortem breakdown of intracrystalline mollusc shell proteins has been extensively investigated, particularly with regard to its potential use as a "molecular clock" for geochronological applications. But despite seventy years of ancient protein research, the fundamental aspects of diagenesis-induced changes to protein structures and sequences remain elusive. In this study we investigate the degradation of intracrystalline proteins by performing artificial degradation experiments on the shell of the thorny oyster, Spondylus gaederopus, which is particularly important for archaeological research. We used immunochemistry and tandem mass tag (TMT) quantitative proteomics to simultaneously track patterns of structural loss and of peptide bond hydrolysis. Powdered and bleached shell samples were heated in water at four different temperatures (80, 95, 110, 140 °C) for different time durations. The structural loss of carbohydrate and protein groups was investigated by immunochemical techniques (ELLA and ELISA) and peptide bond hydrolysis was studied by tracking the changes in protein/peptide relative abundances over time using TMT quantitative proteomics. We find that heating does not induce instant organic matrix decay, but first facilitates the uncoiling of cross-linked structures, thus improving matrix detection. We calculated apparent activation energies of structural loss: E a (carbohydrate groups) = 104.7 kJ/mol, E a (protein epitopes) = 104.4 kJ/mol, which suggests that secondary matrix structure degradation may proceed simultaneously with protein hydrolysis. While prolonged heating at 110 °C (10 days) results in complete loss of the structural signal, surviving peptide sequences were still observed. Eight hydrolysis-prone peptide bonds were identified in the top scoring shell sequence, the uncharacterised protein LOC117318053 from Pecten maximus. Interestingly, these were not the expected "weak" bonds based on published theoretical stabilities calculated for peptides in solution. This further confirms that intracrystalline protein degradation patterns are complex and that the overall microchemical environment plays an active role in protein stability. Our TMT approach represents a major stepping stone towards developing a model for studying protein diagenesis in biomineralised systems., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Accurate predictions of the three-dimensional structures of proteins from their amino acid sequences have come of age. AlphaFold, a deep learning-based approach to protein structure prediction, shows remarkable success in independent assessments of prediction accuracy. A significant epoch in structural bioinformatics was the structural annotation of over 98% of protein sequences in the human proteome. Interestingly, many predictions feature regions of very low confidence, and these regions largely overlap with intrinsically disordered regions (IDRs). That over 30% of regions within the proteome are disordered is congruent with estimates that have been made over the past two decades, as intense efforts have been undertaken to generalize the structure-function paradigm to include the importance of conformational heterogeneity and dynamics. With structural annotations from AlphaFold in hand, there is the temptation to draw inferences regarding the "structures" of IDRs and their interactomes. Here, we offer a cautionary note regarding the misinterpretations that might ensue and highlight efforts that provide concrete understanding of sequence-ensemble-function relationships of IDRs. This perspective is intended to emphasize the importance of IDRs in sequence-function relationships (SERs) and to highlight how one might go about extracting quantitative SERs to make sense of how IDRs function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The need to make sense of the thousands of genetic variants uncovered every day in terms of pathology or biological mechanism is acute. Many insights into how genetic changes impact protein function can be gleaned if three-dimensional structures of the associated proteins are available. The availability of a highly accurate method of predicting structures from amino acid sequences (e.g. Alphafold2) is thus potentially a great boost to those wanting to understand genetic changes. In this paper we discuss the current state of protein structures known for the human and other proteomes and how Alphafold2 might impact on variant interpretation efforts. For the human proteome in particular, the state of the available structural data suggests that the impact on variant interpretation might be less than anticipated. We also discuss additional efforts in structure prediction that could further aid the understanding of genetic variants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Stunning advances have been achieved in addressing the protein folding problem, providing deeper understanding of the mechanisms by which proteins navigate energy landscapes to reach their native states and enabling powerful algorithms to connect sequence to structure. However, the realities of the in vivo protein folding problem remain a challenge to reckon with. Here, we discuss the concept of the "proteome folding problem"-the problem of how organisms build and maintain a functional proteome-by admitting that folding energy landscapes are characterized by many misfolded states and that cells must deploy a network of chaperones and degradation enzymes to minimize deleterious impacts of these off-pathway species. The resulting proteostasis network is an inextricable part of in vivo protein folding and must be understood in detail if we are to solve the proteome folding problem. We discuss how the development of computational models for the proteostasis network's actions and the relationship to the biophysical properties of the proteome has begun to offer new insights and capabilities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Depression is a global mental disorder disease and greatly threatened human health. Xiaochaihutang (XCHT) has been used successfully in treatment of depression for many years in China, but the mechanism is unclear. Using the chronic unpredictable mild stress (CUMS) mice model of depression, the present study aimed to reveal possible antidepressant mechanisms of XCHT from the perspective of liver by analyzing hepatic proteomics in mice. Bioinformatics analysis identified 31 differentially expressed proteins (DEPs), including 5 upregulated and 26 downregulated proteins, between the CUMS model and XCHT groups. The bile secretion pathway was found by KEGG pathway analysis of these DEPs. Four of the 31 differentially expressed proteins, including 2 active proteins involved in bile secretion, carbonic anhydrase 2 (CA2) and cystic fibrosis transmembrane conductance regulator (CFTR), were selected to verify their genes. Four genes (Cyp7a1, Fxr, Shp and Ntcp) related to bile acid synthesis and transport were further investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Both biochemical tests and gene studies demonstrated that CUMS affected bile acid synthesis and transport, while XCHT regulated this pathway. The results indicated that there may be a potential relationship between CUMS induced depression and hepatic injury caused by increased bile acid, and also provide a novel insight to understand the underlying anti-depression mechanisms of XCHT., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Artificial light at night (ALAN) is a widespread environmental pollutant and stressor. Many nocturnal insects have been shown to experience ALAN stress. However, few studies have been conducted to uncover the mechanism by which nocturnal insects respond to ALAN stress. Previous studies suggest that lysine succinylation (Ksuc) is a potential mechanism that coordinates energy metabolism and antioxidant activity under stressful conditions. Mythimna separata (Walker) (M. separata) is a nocturnal insect that has been stressed by ALAN. In this study, we quantified the relative proteomic Ksuc levels in ALAN-stressed M. separata. Of the 466 identified Ksuc-modified proteins, 103 were hypersuccinylated/desuccinylated in ALAN-stressed moths. The hypersuccinylated/desuccinylated proteins were shown to be involved in various biological processes. In particular, they were enriched in metabolic processes, reactive oxygen species (ROS) homeostasis and the neuromuscular system. Furthermore, we demonstrated that Ksuc might affect moth locomotion by intervening with and coordinating these systems under ALAN stress. These findings suggest that Ksuc plays a vital role in the moth response to ALAN stress and moth locomotion behavior and provide a new perspective on the impact of ALAN on nocturnal insect populations and species communities., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ti-based immobilized metal affinity chromatography (IMAC) nanomaterial has shown high potential in phosphoproteome mass-spectrometric (MS) analysis. However, the limited surface area and poor solubility will greatly restrict its use in phosphoproteome research. To overcome these two key drawbacks, a novel Ti-based IMAC nanomaterial was prepared by Ti-bonded β-cyclodextrin (β-CD) anchored on the surface of carbon nanotubes (CNTs) (denoted as COOH-CNTs-CD-Ti) and successfully applied as a biofunctional adsorbent for selectively enriching trace phosphopeptides. In the selective enrichment process, CNTs provided greater surface area for the absorption of phosphopeptides, while β-CD also offered a greater opportunity for the interaction between phosphopeptides and Ti 4+ . COOH-CNTs-CD-Ti with the aforementioned properities exhibited higher selectivity for phosphopeptides from the standard protein digests, the tryptic digests of nonfat milk and human serum, showing a great selective enrichment capability towards complex biological samples., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The diversity of ubiquitin modifications is immense. A protein can be monoubiquitylated, multi-monoubiquitylated, and polyubiquitylated with chains varying in size and shape. Ubiquitin itself can be adorned with other ubiquitin-like proteins and smaller functional groups. Considering different combinations of post-translational modifications can give rise to distinct biological outcomes, characterizing ubiquitylated proteoforms of a given protein is paramount. In this Opinion, we review recent advances in detecting and quantifying various ubiquitin proteoforms using mass spectrometry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Post-translational modifications (PTMs) of proteins extensively diversify the biological information flow from the genome to the proteome and thus have profound pathophysiological implications. Precise dissection of the regulatory networks of PTMs benefits from the ability to achieve conditional control through external optogenetic or chemogenetic triggers. Genetic code expansion provides a unique solution by allowing for site-specific installation of functionally masked unnatural amino acids (UAAs) into proteins, such as enzymes and enzyme substrates, rendering them inert until rapid activation through exposure to light or small molecules. Here, we summarize the most recent advances harnessing this methodology to study various forms of PTMs, as well as generalizable approaches to externally control nodes-of-interest in PTM networks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ensembles of tandem repeats (TRs) in protein sequences expand rapidly to form domains well suited for interactions with proteins. For this reason, they are relatively frequent. Some TRs have known structures and therefore it is advantageous to predict their presence in a protein sequence. However, since most TRs diverge quickly, their detection by classical sequence comparison algorithms is not very accurate. Previously, we developed a method and a web server that used curated profiles and thresholds for the detection of 11 common TRs. Here we present a new web server (REP2) that allows the analysis of TRs in both individual and aligned sequences. We provide currently precomputed analyses for a selection of 78 UniProt reference proteomes. We illustrate how these data can be used to study the evolution of TRs using comparative genomics. REP2 can be accessed at http://cbdm-01.zdv.uni-mainz.de/~munoz/rep/., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Genome-wide protein-protein interaction (PPI) determination remains a significant unsolved problem in structural biology. The difficulty is twofold since high-throughput experiments (HTEs) have often a relatively high false-positive rate in assigning PPIs, and PPI quaternary structures are more difficult to solve than tertiary structures using traditional structural biology techniques. We proposed a uniform pipeline, Threpp, to address both problems. Starting from a pair of monomer sequences, Threpp first threads both sequences through a complex structure library, where the alignment score is combined with HTE data using a naïve Bayesian classifier model to predict the likelihood of two chains to interact with each other. Next, quaternary complex structures of the identified PPIs are constructed by reassembling monomeric alignments with dimeric threading frameworks through interface-specific structural alignments. The pipeline was applied to the Escherichia coli genome and created 35,125 confident PPIs which is 4.5-fold higher than HTE alone. Graphic analyses of the PPI networks show a scale-free cluster size distribution, consistent with previous studies, which was found critical to the robustness of genome evolution and the centrality of functionally important proteins that are essential to E. coli survival. Furthermore, complex structure models were constructed for all predicted E. coli PPIs based on the quaternary threading alignments, where 6771 of them were found to have a high confidence score that corresponds to the correct fold of the complexes with a TM-score >0.5, and 39 showed a close consistency with the later released experimental structures with an average TM-score = 0.73. These results demonstrated the significant usefulness of threading-based homologous modeling in both genome-wide PPI network detection and complex structural construction., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human mitochondria are complex and highly dynamic biological systems, comprised of over a thousand parts and evolved to fully integrate into the specialized intracellular signaling networks and metabolic requirements of each cell and organ. Over the last two decades, several complementary, top-down computational and experimental approaches have been developed to identify, characterize and modulate the human mitochondrial system, demonstrating the power of integrating classical reductionist and discovery-driven analyses in order to de-orphanize hitherto unknown molecular components of mitochondrial machineries and pathways. To this goal, systematic, multiomics-based surveys of proteome composition, protein networks, and phenotype-to-pathway associations at the tissue, cell and organellar level have been largely exploited to predict the full complement of mitochondrial proteins and their functional interactions, therefore catalyzing data-driven hypotheses. Collectively, these multidisciplinary and integrative research approaches hold the potential to propel our understanding of mitochondrial biology and provide a systems-level framework to unraveling mitochondria-mediated and disease-spanning pathomechanisms., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nascent proteome presents dynamic changes in response to a certain stimulus. Thus, monitoring nascent proteome is critical to uncovering the involved biological mechanism. But the low-abundance of nascent proteome against an overwhelming pre-existing proteome limits its identification and quantification. Herein, we present a novel strategy to enrich nascent proteome from whole cell lysate for further analysis by mass spectrometry. We employed a terminal alkyne and disulfide functionalized agarose resin to capture nascent proteome which had been labeled by L-azidohomoalanine. Results from the western blot, silver staining and pulse metabolic labeling suggested that the nascent proteome could be enriched efficiently. Applied to Hela cells, the method identified about 700 nascent proteins with good correlation with previous reports. The above indicates that our strategy can be used to reveal the proteome dynamics of biological processes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In the present study, a tandem mass tag-labeled quantitative proteomic analysis was performed to compare the differences in protein profiles between Tibetan pig (TP) tenderloin and Yorkshire pig (YP) tenderloin. A total of 171 proteins were identified as differentially abundant proteins (DAPs) from 1448 quantified proteins. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathway analyses revealed that upregulated DAPs in the TP tenderloin were mainly involved in energy production, muscle contraction, immunity and defense, while downregulated DAPs were mainly involved in glutathione metabolism. These DAPs and the pathways they participate in were related to meat characteristics. TP tenderloin has a deeper color and higher pH, but lower water content and cooking loss. This study provides insights into the mechanism underlying the differences in protein profiles between TP and YP tenderloins. The findings can provide a better understanding of the formation mechanism for Tibetan pork edible quality., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In Nanomedicine, carbon-based nanomaterials, like Carbon Nanotubes (CNT), are considered potential candidates as drug delivery systems. In vivo adsorption of physiological proteins onto carbon nanotubes, through noncovalent interactions, forms a protein corona or bio corona, able to influence biological properties and biocompatibility of CNT. This study aimed to explore the composition of protein corona formed onto PEGylated Multi-Walled Carbon Nanotubes (MWCNT-PEG5k), after their incubation in human plasma. Plasma proteins were sequentially eluted in different conditions by using both native and denaturant buffers, useful to characterize soft and hard corona. Proteomic methods and mass spectrometry analysis have identified proteins in soft corona, involved in the regulation of immune response and in the CNT transport, and biomolecules in hard corona with a role in the maintenance of host homeostasis. These promising results have demonstrated the potential of PEGylated Multi-Walled Carbon Nanotubes as future candidates for drug delivery., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Shotgun proteomics is a high-throughput technology which has been developed with the aim of investigating the maximum number of proteins in cells in a given experiment. However, protein discovery and data generation vary in depth and coverage when different technical strategies are selected. In this study, three different sample preparation approaches, and peptide or protein fractionation methods, were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), filter-aided sample preparation coupled with gas phase fractionation (FASP-GPF), and FASP - high pH reversed phase fractionation (HpH). Fractions were initially analyzed and compared using nanoflow liquid chromatography - tandem mass spectrometry (nanoLC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and analytical replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time. A second set of experiments was performed, comparing FASP-GPF, SDS-PAGE and FASP-HpH using a Q Exactive Orbitrap mass spectrometer. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a substantial increase in protein identifications, and an even greater increase in peptide identifications. This shows that the main advantage of using the higher resolution instrument is in increased proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches, when applied to this type of sample., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Geographical variation of animal venom is common among venomous animals. This kind of intraspecific variation based on geographical location mainly concerned from envenomation cases and brought new problems in animal venom studies, including venom components regulatory mechanisms, differentiation of venom activities, and clinical treatment methods. At present, food is considered as the most related factor influencing venom development. Related research defined the variational venomous animal species by the comparison of venom components and activities in snakes, jellyfish, scorpions, cone snails, ants, parasitoid wasps, spiders and toads. In snake venom studies, researchers found that antivenom effectiveness was variated to different located venom samples. As described in some snake venom research, developing region-specific antivenom is the development trend. The difficulties of developing region-specific antivenom and theoretical solutions have been discussed. This review summarized biological studies of animal venom geographical variation by species, compared venom components and major biological activities of the vary venom from the same species, and listed the basic methods in comparing venom protein compositions and major toxicity differences to provide a comprehensive reference., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion channels and membrane receptors, and platelet aggregation and platelet plug formation. Toxins target these effectors and effects at high affinity and selectivity. From a pharmacological perspective, snake venom compounds are a valuable resource for drug discovery and development. However, a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures. Getting molecular information from complex mixtures such as snake venoms requires proteomic analyses, generally combined with transcriptomic analyses of venom glands. The present review summarizes current knowledge and highlights important recent advances in venomics with special emphasis on contemporary separation techniques and bioinformatics that have begun to elaborate the complexity of snake venoms. Several analytical techniques such as two-dimensional gel electrophoresis, RP-HPLC, size exclusion chromatography, ion exchange chromatography, MALDI-TOF-MS, and LC-ESI-QTOF-MS have been employed in this regard. The improvement of separation approaches such as multidimensional-HPLC, 2D-electrophoresis coupled to soft-ionization (MALDI and ESI) mass spectrometry has been critical to obtain an accurate picture of the startling complexity of venoms. In the case of bioinformatics, a variety of software tools such as PEAKS also has been used successfully. Such information gleaned from venomics is important to both predicting and resolving the biological activity of the active components of venoms, which in turn is key for the development of new drugs based on these venom components., (Published by Elsevier B.V.)

Atlantic salmon is often adulterated or substituted by rainbow trout with much lower price and quality. However, it is extremely difficult to distinguish Atlantic salmon and rainbow trout due to their similar appearance and close relationship in species. In the present work, untargeted and targeted proteomics approaches were both implemented to identify species-specific peptide biomarkers of Atlantic salmon and rainbow trout. Potential peptide biomarkers were obtained through matching HRMS data with UniProt database, screened by BLAST and then verified with real samples. Five peptide biomarkers were identified each for Atlantic salmon and rainbow trout. MRM method was established for quantitative measurement of rainbow trout Adulteration in Atlantic salmon, showing high sensitivity and repeatability. The biomarker peptide GDPGPGGPQGEQGVVGPAGISGDK was used for quantification. The limit of the detection (LOD) of adulteration of rainbow trout is 0.19%, and the limit of quantitation (LOQ) is 0.62%. Furthermore, this method was successfully applied to analyze a number of Atlantic salmon and Rainbow trout samples from different regions and different batches, as well as commercially available processed products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Traditional approaches in the development of small-molecule drugs typically aim to inhibit the biochemical activity of functional protein domains. In contrast, targeted protein degradation aims to reduce overall levels of disease-relevant proteins. Mechanistically, this can be achieved via chemical ligands that induce molecular proximity between an E3 ubiquitin ligase and a protein of interest, leading to ubiquitination and degradation of the protein of interest. This paradigm-shifting pharmacology promises to address several limitations inherent to conventional inhibitor design. Most notably, targeted protein degradation has the potential not only to expand the druggable proteome beyond the reach of traditional competitive inhibitors but also to develop therapeutic strategies of unmatched selectivity. This review briefly summarizes key challenges that remain to be addressed to deliver on these promises and to realize the full therapeutic potential of pharmacologic modulation of protein degradation pathways., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: There is a growing body of evidence supporting the role of whole-body cryostimulation (WBC) and sauna - bathing as treatments for relaxation, mental well-being and several health problems. Despite their polar opposite temperatures, both of these treatments come with a dose of similar health benefits. This study is designed to compare effects of WBC and sauna application on the athletes' response to exercise., Design: The blood samples were collected from 10 professional cross-country skiers at four stages: before exercise, after exercise, at 1-h recovery and after 24 h of rest in sessions before and after 10 thermal treatments. Differential scanning calorimetry (DSC) was used to examine the process of serum denaturation. The parameters of endothermic transition were compared at various stages of each exercise session., Results: Post-exercise changes in DSC profiles of athlete's blood serum are similar in character but clearly stronger in the session held after sauna treatments and slightly weaker after WBC than those in the session not preceded by treatments. These changes can be, at least in part, explained by the exercise induced increase in the concentration of oxidized albumin. A return of serum denaturation transition to pre-exercise shape has been observed within a few hours of rest. It suggests relatively quick restoration of a fraction of non-oxidized albumin molecules during the recovery period., Conclusions: An exercise performed by athletes after a series of sauna treatments leads to temporary greater modification of the blood serum proteome than the similar exercise during the session preceded by WBC treatments., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aims: Nonhuman primates have been used to investigate pathogenic mechanisms and evaluate immune responses following Chlamydia trachomatis inoculation. This study aimed to systemically profile antibody responses to C. trachomatis infection in nonhuman primates., Materials and Methods: Sera were obtained from 4 pig-tailed and 8 long-tailed macaques which were intravaginally or ocularly infected with live C. trachomatis organisms, and analyzed by C. trachomatis proteome array of antigens., Key Findings: The sera from 12 macaques recognized total 172 C. trachomatis antigens. While 84 antigens were recognized by pig-tailed macaques intravaginally infected with serovar D strain, 125 antigens were recognized by long-tailed macaques ocularly infected with serovar A, and 37 antigens were recognized by both. Ocular inoculation with virulent A2497 strain induced antibodies to more antigens. Among the antigens uniquely recognized by A2497 strain infected macaques, outer membrane complex B antigen (OmcB) induced robust antibody response. Although macaques infected by less virulent A/HAR-13 strain failed to develop antibodies to OmcB, reinfection by A2497 strain induced high levels of antibodies to OmcB., Significance: Proteome array has revealed a correlation of chlamydial infection invasiveness with chlamydial antigen immunogenicity, and identified antibody responses to OmcB potentially as biomarkers for invasive infection with C. trachomatis., Competing Interests: Declaration of competing interest All authors have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The venom protein components of Malabar pit viper (Trimeresurus malabaricus) were identified by combining SDS-PAGE and ion-exchange chromatography pre-fractionation techniques with LC-MS/MS incorporating Novor and PEAKS-assisted de novo sequencing strategies. Total 97 proteins that belong to 16 protein families such as L-amino acid oxidase, metalloprotease, serine protease, phospholipase A 2 , 5'-nucleotidase, C-type lectins/snaclecs and disintegrin were recognized from the venom of a single exemplar species. Of the 97 proteins, eighteen were identified through de novo approaches. Immunological cross-reactivity assessed through ELISA and western blot indicate that the Indian antivenoms binds less effectively to Malabar pit viper venom components compared to that of Russell's viper venom. The in vitro cell viability assays suggest that compared to the normal cells, MPV venom induces concentration dependent cell death in various cancer cells. Moreover, crude venom resulted in chromatin condensation and apoptotic bodies implying the induction of apoptosis. Taken together, the present study enabled in dissecting the venom proteome of Trimeresurus malabaricus and revealed the immuno-cross-reactivity profiles of commercially available Indian polyvalent antivenoms that, in turn, is expected to provide valuable insights on the need in improving antivenom preparations against its bite., Competing Interests: Declaration of competing interest Nil., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A microfluidic platform based on the integration of denaturation and online immobilized enzyme reactor (IMER) digestion for protein pretreatment was first developed on a glass chip. The design of three inlet channels and two groups of snake channel in glass chip can allow the protein solution, the reducing reagent and the alkylating agent to be simultaneously injected into the chip channel and ensured the reaction solution on-line efficient mixing and sufficient reacting. By thiol-ene click chemistry, the capillary-based and glass chip-based trypsin IMER on the surface of poly(trimethylolpropane trimethacrylate) monolith were fabricated. The wide range of flow rate tolerance (0.8-5.0 μL/min), and the acceptable reproducibility (RSD% = 3.1%, n = 5) and stability (13.8% decrease of enzyme activity in 2 months) indicated the feasibility of using IMER for online digestion of proteins. Compared with the solution denaturation-offline IMER digestion, the integrated microfluidic platform of chip denaturation-chip IMER and chip denaturation-online IMER have comparable protein identification ability for mouse liver protein with a similar number of protein (798 or 826 vs. 843) and unique peptides (3923 or 4593 vs. 3916). More importantly, the easy and fast digestion of protein samples and possible combination with MS revealed that this microfluidic platform can be a potential method for rapid proteomics analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

For mass spectrometry (MS)-based N-glycoproteomics, selective enrichment of N-glycopeptides prior to MS analysis is a crucial step to reduce sample complexity. Enrichment based on covalent coupling is as an increasingly attractive strategy due to the unbiased and highly specific features. However, most of current covalent coupling reactions for N-glycopeptides enrichment are still limited by long coupling time and harsh coupling conditions. Herein, we developed a thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome. With the use of facile synthesis of Cys-terminated magnetic nanoparticles, the oxidized glycan moieties on glycopeptides could be selectively captured by the β-amino thiols groups on the surface of magnetic nanoparticles through thiazolidine formation. The coupling could be achieved within 30 min under mild condition, eliminating the addition of toxic catalyst or sample-destroying reducing agent. Also, the great enrichment performance for N-glycopeptides were obtained in terms of sensitivity (low fmol levels), selectivity (extracting N-glycopeptides from the mixture of glycopeptides and non-glycopeptides at a 1:100 molar ratio) and reproducibility (CVs<26%). Finally, this proposed method was successfully demonstrated by analyzing the N-glycoproteome from 2 μL human serum, which offers an alternative purification method for analysis of N-glycoproteome from complex biological samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Small molecule metabolites play important roles in regulating protein functions, which are acted through either covalent non-enzymatic post-translational modifications or non-covalent binding interactions. Chemical proteomic strategies can help delineate global landscapes of cellular protein-metabolite interactions and provide molecular insights about their mechanisms of action. In this review, we summarized the recent progress in developments and applications of chemoproteomic strategies to profile protein-metabolite interactions., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Protein-RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic-aqueous phase separation-based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein-RNA interactions., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Understanding the molecular mechanisms of endogenous and environmental metabolites is crucial for basic biology and drug discovery. With the genome, proteome, and metabolome of many organisms being readily available, researchers now have the opportunity to dissect how key metabolites regulate complex cellular pathways in vivo. Nonetheless, characterizing the specific and functional protein targets of key metabolites associated with specific cellular phenotypes remains a major challenge. Innovations in chemical biology are now poised to address this fundamental limitation in physiology and disease. In this review, we highlight recent advances in chemoproteomics for targeted and proteome-wide analysis of metabolite-protein interactions that have enabled the discovery of unpredicted metabolite-protein interactions and facilitated the development of new small molecule therapeutics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intrinsically disordered proteins lack a stable three-dimensional structure under physiological conditions. While this property has gained considerable interest within the past two decades, disorder poses substantial challenges to experimental characterization efforts. In effect, numerous computational tools have been developed to predict disorder from primary sequences, however, interpreting the output of these algorithms remains a challenge. To begin to bridge this gap, we present Disorder Atlas, web-based software that facilitates the interpretation of intrinsic disorder predictions using proteome-based descriptive statistics. This service is also equipped to facilitate large-scale systematic exploratory searches for proteins encompassing disorder features of interest, and further allows users to browse the prevalence of multiple disorder features at the proteome level. As a result, Disorder Atlas provides a user-friendly tool that places algorithm-generated disorder predictions in the context of the proteome, thereby providing an instrument to compare the results of a query protein against predictions made for an entire population. Disorder Atlas currently supports ten eukaryotic proteomes and is freely available for non-commercial users at http://www.disorderatlas.org., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A rapid and efficient method to isolate global N-termini is presented. Utilizing laser-assisted proteolysis and Fe 3 O 4 microsphere, protein N-termini could be isolated in 4 h. The amino-blocked protein was digested by trypsin assisted by laser radiation, shortening the digest time from overnight to 40 s. Non-N-terminal peptides were characterized by a tryptic free amino in their N-term, which could be derived with sulfhydryl by traut' s reagent efficiently and then coupled with Fe 3 O 4 microspheres nearly completely in less than 4 h. The rapid method was beneficial for the identification of unstable N-termini in short-lived proteins. Human serum albumin was studied as a model. The N-terminus was successfully isolated from the digest within 4 h. Also, 2011 N-terminal peptides out of 936 proteins in mouse liver proteome sample were identified using liquid chromatography-tandem mass spectrometer (LC-MS/MS). This method was demonstrated as a facile and efficient N-termini enrichment method for targeted protein N-termini analysis, especially those with short half-life., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We present a method to preserve and process urine proteins for proteomic analysis in a filter aided sample preparation (FASP) format. The method combines concentration of urine proteins in ultrafiltration devices, their thermal stabilization, allowing long term storage of the samples, and filter aided sample preparation. Proteomes of four different urines were preserved during 48 h and 6 months using (i) the classic freeze preservation at -20 °C, (ii) snap-heated freeze-free preservation at laboratory temperature (20 °C) and (iii) snap-heated preservation at -60 °C. The three storage methods can effetely preserve the urine proteome for at least 6 months without significant alterations. Abundances of more than 500 proteins and specially 24 selected -cleared or -approved protein assayed in serum or plasma were found similar within the three preservation methods assessed. The new method here proposed dramatically simplifies the conditions for preserving the urine proteome for biobanks in terms of space and storage, including lowering the risks of sample degradation caused by misfunction of the freezer. Furthermore, the shipping of large number of samples can be made without the need of freezing. The application of the FASP format to isolate and preserve the proteins facilitates long-term storage and processing of proteome of urine samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background & Aims: The aim of the paper is to investigate the effect of the active fraction extracted from the Xinjiang Bactrian camel whey on the human cancer cells using an in vitro and in vivo model of human carcinoma of the esophagus., Methods and Results: Our results demonstrated that an antitumor active fraction, TR35, isolated from Xinjiang Bactrian camel milk could significantly inhibit Eca109 cell proliferation and induce its apoptosis (indicated by MTT assay, Annexin V-FITC Apoptosis Detection, and caspase-3 activity). Moreover, we found that TR35 could inhibit the growth of xenografted tumor in nude mice without loss in body weight. Furthermore, we used RNA-Seq and 2-DE combined Mass Spectrometry analysis to identify differentially expressed RNA and protein markers of apoptosis and necrosis. Compared with untreated Eca109 cells, a total of 405 differentially expressed genes and 55 differentially expressed proteins were identified in TR35 treated Eca109 cells. KEGG analysis uncovered signaling pathways closely associated with cancer inhibition that were enriched in the TR35-treated cells., Conclusions: These results might implicate that downregulation of specific proteins identified in this study may be the cause of this tumor growth inhibition. This study sheds light on the potential therapeutic advantages based on the historical anti-cancer activities of camel milk., (Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Objective: Major depression (MDD) and a lifetime history of MDD are characterized by increased nitrosylation, while bipolar disorder type 1 (BP1), but not BP2, is accompanied by highly increased levels of oxidative stress and nitric oxide (NO) production. Nevertheless, it is unknown whether nitrosylation is involved in BP and whether there are differences in nitrosylation between BP1 and BP2., Methods: Serum IgM antibodies directed against nitroso (NO)-adducts were examined in MDD, BP1, BP2 and healthy controls, namely IgM responses to NO-cysteine, NO-tryptophan (NOW), NO-arginine and NO-albumin (SBA) in association with IgA/IgM responses to LPS of Gram-negative bacteria, IgG responses to oxidized low-density lipoprotein (ox-LDL) and serum peroxides., Results: Serum IgM levels against NO adducts were significantly higher in BP1 and MDD as compared with healthy controls, whereas BP2 patients occupied an intermediate position. IgM responses to NO-albumin were significantly higher in BP1 and MDD than in BP2 patients. There were highly significant associations between the IgM responses to NO-adducts and IgG responses to ox-LDL and IgA/IgM responses to Gram-negative bacteria., Conclusions: BP1 and MDD are characterized by an upregulation of the nitrosylome (the proteome of nitrosylated proteins) and increased IgM responses to nitrosylated conjugates. Increased nitrosylation may be driven by increased bacterial translocation and is associated with lipid peroxidation processes. Innate-like (B1 and marginal zone) B cells and increased nitrosylation may play a key role in the major affective disorders through activation of immune-inflammatory and oxidative pathways, cardiovascular comorbidity and impairments in antioxidant defenses, neuro-glial interactions, synaptic plasticity, neuroprotection, neurogenesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Snake venoms are complex mixtures of a large number of distinct proteins and peptides with biological activity. Peptide spectral libraries are compilations of previously identified MS/MS spectra obtained from proteomics experiments. Here we present the generation and use of a Venom Peptidome and a Venom Proteome spectral library for the analysis of venom proteomes and peptidomes from distinct snake species., (Copyright © 2019 Elsevier Inc. All rights reserved.)

On average a human cell type expresses around 10,000 different protein coding genes synthesizing all the different molecular forms of the protein product (proteoforms) found in a cell. In a typical shotgun bottom up proteomic approach, the proteins are enzymatically cleaved, producing several 100,000 s of different peptides that are analyzed with liquid chromatography-tandem mass spectrometry (LC-MSMS). One of the major consequences of this high sample complexity is that coelution of peptides cannot be avoided. Moreover, low abundant peptides are difficult to identify as they have a lower chance of being selected for fragmentation due to ion-suppression effects and the semi-stochastic nature of the precursor selection in data-dependent shotgun proteomic analysis where peptides are selected for fragmentation analysis one-by-one as they elute from the column. In the current study we explore a simple novel approach that has the potential to counter some of the effect of coelution of peptides and improves the number of peptide identifications in a bottom-up proteomic analysis. In this method, peptides from a HeLa cell digest were eluted from the reverse phase column using three different elution solvents (acetonitrile, methanol and acetone) in three replicate reversed phase LC-MS/MS shotgun proteomic analysis. Results were compared with three technical replicates using the same solvent, which is common practice in proteomic analysis. In total, we see an increase of up to 10% in unique protein and up to 30% in unique peptide identifications from the combined analysis using different elution solvents when compared to the combined identifications from the three replicates of the same solvent. In addition, the overlap of unique peptide identifications common in all three LC-MS analyses in our approach is only 23% compared to 50% in the replicates using the same solvent. The method presented here thus provides an easy to implement method to significantly reduce the effects of coelution and ion suppression of peptides and improve protein coverage in shotgun proteomics. Data are available via ProteomeXchange with identifier PXD011908., (Copyright © 2019. Published by Elsevier B.V.)

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD., (Copyright © 2019. Published by Elsevier B.V.)

The oviduct provides the optimal micro milieu for early embryo development. However, accessing the bovine oviductal fluid in vivo for analysis is still challenging and therefore the oviductal fluid is usually collected post mortem. In the study presented here we introduce a novel approach to gain minimal invasive access to the bovine oviductal fluid proteome in vivo by transvaginal endoscopy at different stages of the estrous cycle. The first experiment aimed at transferring C4 derivatised magnetic beads to bind the oviductal fluid proteome in situ. Protein carrying beads were recovered by flushing the oviduct and proteins were eluted. In the second experiment a flushing solution was injected into and aspirated from the oviduct repeatedly. The flushing solution was centrifuged to separate the fluid from the cellular debris. Proteins were identified by nano-LC-MS/MS. Two different stages of the estrous cycle (Day 1 and Day 3) were analyzed in samples from 30 heifers. Both methods were applied successfully and in total, more than 3000 proteins were identified, so far representing the most comprehensive OF proteome published. This new minimal invasive approach to access the bovine oviductal fluid proteome facilitates future innovative experimental designs to study the role of the oviductal micro environment during early embryo development., (Copyright © 2019 Elsevier Inc. All rights reserved.)