Bottom-up Proteomic Approach Research Articles

Proteomic Characterization of Colorectal Cancer Cells versus Normal-Derived Colon Mucosa Cells: Approaching Identification of Novel Diagnostic Protein Biomarkers in Colorectal Cancer.

In the western world, colorectal cancer (CRC) is the third most common cause of cancer-related deaths. Survival is closely related to the stage of cancer at diagnosis striking the clinical need for biomarkers capable of early detection. To search for possible biological parameters for early diagnosis of CRC we evaluated protein expression for three CREC (acronym: Cab45, reticulocalbin, ERC-55, calumenin) proteins: reticulocalbin, calumenin, and ERC-55 in a cellular model consisting of a normal derived colon mucosa cell line, NCM460, and a primary adenocarcinoma cell line of the colon, SW480. Furthermore, this cellular model was analyzed by a top-down proteomic approach, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for novel putative diagnostic markers by identification of differentially expressed proteins between the two cell lines. A different colorectal carcinoma cell line, HCT 116, was used in a bottom-up proteomic approach with label-free quantification (LFQ) LC–MS/MS. The two cellular models gave sets of putative diagnostic CRC biomarkers. Various of these novel putative markers were verified with increased expression in CRC patient neoplastic tissue compared to the expression in a non-involved part of the colon, including reticulocalbin, calumenin, S100A6 and protein SET. Characterization of these novel identified biological features for CRC patients may have diagnostic potential and therapeutic relevance in this malignancy characterized by a still unmet clinical need.

Venomics of the Central American Lyre Snake Trimorphodon quadruplex (Colubridae: Smith, 1941) from Costa Rica

Rear-fanged colubrid snakes include hundreds of species globally that possess a Duvernoy's venom gland and often one-several enlarged rear maxillary teeth. We investigated the venom proteome of the Central American Lyre Snake (Trimorphodon quadruplex), a moderate-sized rear-fanged colubrid snake and the southernmost Trimorphodon, using a bottom-up proteomic approach coupled with enzyme and inhibitor assays, cytotoxicity assays and lethal toxicity assays. Several enzymes uncommonly observed in colubrid venoms were purified and characterized further. Trimorphodon quadruplex has a rather low complexity venome, typical of many rear-fanged snakes, but its venom contains L-amino acid oxidase, phospholipase A2, and a dimeric 3FTx, and 3FTxs dominate the proteome. Its PLA2 is catalytically quite active, but it lacks myotoxicity or acute toxicity; LAAO exhibits conserved structure and appears to be highly labile. Several P-III metalloproteinases are present and hydrolyze azocasein and the α-subunit of fibrinogen but lack hemorrhagic activity. Trimorphodon quadruplex produces venom and retains constriction, utilizing both chemically-mediated and mechanical feeding modes. SignificanceWe demonstrate that T. quadruplex venom proteins are similar to those found in front-fanged snake species are present but show different biological activities. Our results underscore the importance of considering the biological roles of venoms from more than a mammal-centric perspective.

Quality over quantity: A qualitative, targeted bottom-up proteomics approach to genotyping apolipoprotein L1

A targeted, bottom-up proteomic assay was developed for the qualitative detection of apolipoprotein L1 (ApoL1) protein variants (G0, G1, and G2) in blood plasma for identification of high and low renal risk genotypes. Following trypsin digestion of liquid or dry plasma, surrogate peptides specific to each ApoL1 variant were detected by liquid chromatography-tandem mass spectrometry along with two surrogate peptides common among all variants which served as internal (positive) controls to verify correct sample processing. Using isotopically labeled peptide internal standards, the presence or absence of each surrogate peptide was determined using multiple objective metrics including: 1) retention time confirmation relative to its internal standard, 2) comparison of the internal standard-normalized response relative to pre-established thresholds for confident detection, and 3) ion ratio monitoring. Based on the pattern of variant-specific surrogate peptides detected, the genotype was accurately inferred. The final, optimized method was fully validated for liquid plasma specimens, as well as dry plasma specimens collected on a laminar flow blood separation device. For both specimen types, the latter which can be self-collected for remote or in-home sampling, the assay was shown to be reproducible, interference-free with the exception of gross hemolysis, and accurate relative to Sanger sequencing (100% agreement). This targeted, qualitative bottom-up proteomic assay for detection of ApoL1 variants in blood provides a high-throughput, cost-effective alternative to molecular methods and has potential implications to improve organ allocation by facilitating screening kidney donors for high-risk ApoL1 genotypes, but could be applicable for genotyping other clinically relevant blood proteins variants.

Bottom-up Structural Proteomics: Cryo-EM of Protein Complexes Enriched from the Cellular Milieu

X-ray crystallography and recombinant protein production have enabled an exponential increase in atomic structures, but often require non-native constructs involving mutations or truncations, and are challenged by membrane proteins and large multi-component complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic resolution cryoEM maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria parasite Plasmodium falciparum, an organism that has resisted traditional structural biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach opens the door to atomic structures of previously intractable biological systems.

Shotgun proteomics, in-silico evaluation and immunoblotting assays for allergenicity assessment of lesser mealworm, black soldier fly and their protein hydrolysates

Since 2018, insects have belonged the category of Novel Foods and the presence of allergens represents one of the main hazards connected to their consumption, also due to the potential cross-reactivity with Arthropoda pan-allergens. In the present work, the allergenicity assessment of black soldier fly and lesser mealworm was performed with a shotgun bottom-up proteomic approach combined with in-silico assessment, followed by IgG- and IgE-immunoblotting experiments. The peptides identified, filtered for their abundance and robustness, belonged mainly to muscle proteins, which represented the most abundant protein group. The relevant potential allergens were in-silico identified by sequence similarity to known allergens, and among them tropomyosin resulted the most abundant insect allergen. IgG-immunoblotting analysis with anti-Tropomyosin I antibodies and IgE-immunoblotting assay with serum from patient allergic to crustacean tropomyosin were performed in order to assess the immunoreactivity in both insects. The immunoassays were carried out also on protein hydrolysates extracted by treating insects with Protease from Bacillus licheniformis (1%, 60 °C, pH 7.5). While IgG-immunoblotting demonstrated the loss of immunoreactivity for both hydrolysates, IgE-immunoblotting showed a partial immunoreactivity preservation, also after hydrolysis, in the case of black soldier fly hydrolysate, and a total loss of immunoreactivity for lesser mealworm hydrolysate

Impact of an Antifungal Insect Defensin on the Proteome of the Phytopathogenic Fungus Botrytis cinerea.

ETD151, an analogue of the antifungal insect defensin heliomicin, is an antifungal peptide active against yeasts and filamentous fungi. To decipher the mechanisms underlying its molecular action on the phytopathogenic fungus Botrytis cinerea, a necrotrophic pathogen responsible for gray mold disease, we investigated the changes in 3 day-old mycelia upon treatment with different concentrations of ETD151. Optical and fluorescence microscopies were used prior to establishing the peptide/protein profiles through two mass spectrometry approaches: MALDI profiling, to generate molecular mass fingerprints as peptide signatures, and a gel-free bottom-up proteomics approach. Our results show that a concentration of ETD151 above the half-maximal inhibitory concentration can alter the integrity of the mycelial structure of B. cinerea. Furthermore, reproducible modifications of the peptide/protein composition were demonstrated in the presence of ETD151 within a 1500-16,000 mass (m/z) range. After the robustness of LC-ESI-MS/MS analysis on B. cinerea mycelial extracts was confirmed, our analyses highlighted 340 significantly modulated proteins upon treatment with ETD151 within a 4.8-466 kDa mass range. Finally, data mapping on KEGG pathways revealed the molecular impact of ETD151 on at least six pathways, namely, spliceosome, ribosome, protein processing in endoplasmic reticulum, endocytosis, MAPK signaling pathway, and oxidative phosphorylation.

A Procedure for Analyzing the Proteomic Proteomics Profile of Schistosoma mansoni Cercariae.

Schistosomiasis is one of the most important helminthic parasitic infections in the world, with over 700million people at risk of infection. Species of Schistosoma have a complex life cycle involving the infection of freshwater snails before infecting their mammalian definitive host. Taking about 130,000 lives per annum, S. mansoni is the major cause of intestinal schistosomiasis worldwide. Within Biomphalaria glabrata snails, asexual replication of the parasite gives rise to cercariae larvae. Cercariae actively penetrate the host's skin to complete their life cycle and eventually transform into adult worms. If left untreated, intestinal schistosomiasis can lead to peripheral destruction of the portal vein system, gastric hemorrhage from esophageal varices, as well as hepatic failure. Mass spectrometry (MS) is the method of choice for proteomics analysis. The bottom-up proteomics approach-also known as "shotgun proteomics"-typically includes a protein extraction and solubilization step followed by proteolytic digestion and tandem MS (MS/MS) analysis. Proteins are later identified by peptide de novo sequencing upon MS and MS/MS spectra of digest peptides. In this chapter, we introduce an analytical workflow for proteome profiling of S. mansoni cercariae using bottom-up proteomics. The cercariae were isolated and lysed. Proteins were then extracted, enzymatically digested, and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins were identified using MaxQuant software. Cercariae are the first life stage of the parasite S. mansoni which humans encounter, and conducting proteomic analysis on this life cycle stage can shed light on possible drug or vaccine candidates to help disable the parasite's ability to infect or arm the immune system for parasite clearance.

11: Proteomics Analysis of biological compartments in pregnancies complicated by intra-amniotic infection/inflammation (Triple-I) and preterm birth

Triple I is an important cause of neonatal morbidity and mortality. A relevant clinical question is which type of biological sample [amniotic fluid (AF), maternal blood (MB), cord blood (CB), placenta (PL) and fetal membranes (FM)] reflects the best presence of Triple I. Here, we present a simultaneous, systematic and unbiased study of biological compartments in women with Triple I using a bottom-up proteomics approach. AF, MB, CB, and protein lysates of FM and PL from 4 women with Triple I (GA: 28±1 wks) and 5 women (GA: 31±1 wks, p=.07) with idiopathic PTB (CRL). All biological samples were albumin depleted, tryptic digested and subjected to state-of-art mass spectrometry by using a Velos Pro mass spectrometer coupled to a PepMap Easy-Spray C18 column on a 1D nano Acquity UPLC. Spectra were searched through Proteome Discoverer 1.4 with SEQUEST against Uniprot human database. Protein network analysis was done utilizing Cytoscape software platform (V.3.7.1) with STRING and DyNet Analyzer. With a cutoff of 2 for peptide spectra matching, 1,126 proteins were identified in at least one compartment. The proteins were gene enriched to 708 nodes and 4,904 edges (confidence level 0.90) for all the compartments of Triple I patients (Fig A). The AF had the top dynamic Protein-Protein-Interaction network confirming AF harbors most biomarkers discriminative of Triple I. FM demonstrated the second most varied network after AF, while other compartments remained less perturbed. In the AF the most varying genes clusters related to antibacterial and immune pathways. Among them, histones and keratins constituted two distinctive networks with least heterogeneity. Compared with CRL patients the keratin cluster from Triple I patients displayed a shift between inflammation barrier to tissue repair (Fig B). Our novel analysis suggests that AF is the most relevant biological sample to identify Triple I. In Triple I there is a shift between fetal inflammation to tissue repair, which cannot entirely be identified through the analysis of FM, MB, CB or PL.

Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu.

X-ray crystallography and recombinant protein production have enabled an exponential increase in atomic structures, but often require non-native constructs involving mutations or truncations, and are challenged by membrane proteins and large multi-component complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic resolution cryoEM maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria parasite Plasmodium falciparum, an organism that has resisted conventional structural biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson's Disease.

Parkinson’s disease (PD) is the second most frequent neurodegenerative disease worldwide and the availability of early biomarkers and novel biotargets represents an urgent medical need. The main pathogenetic hallmark of PD is the specific loss of nigral dopaminergic neurons, in which mitochondrial dysfunction plays a crucial role. Mitochondrial proteases are central to the maintenance of healthy mitochondria and they have recently emerged as drug targets. However, an exhaustive characterization of these enzymes and their targets is still lacking, due to difficulties in analyzing proteolytic fragments by bottom-up proteomics approaches. Here, we propose the “mitochondrial dimethylation-TAILS” strategy, which combines the isolation of mitochondria with the enrichment of N-terminal peptides to analyze the mitochondrial N-terminome. We applied this method in a cellular model of altered dopamine homeostasis in neuroblastoma SH-SY5Y cells, which recapitulates early steps of PD pathogenesis. The main aim was to identify candidate mitochondrial proteases aberrantly activated by dopamine dysregulation and their cleaved targets. The proposed degradomics workflow was able to improve the identification of mitochondrial proteins if compared to classical shotgun analysis. In detail, 40% coverage of the mitochondrial proteome was obtained, the sequences of the transit peptides of two mitochondrial proteins were unveiled, and a consensus cleavage sequence for proteases involved in the processing of mitochondrial proteins was depicted. Mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD013900. Moreover, sixty-one N-terminal peptides whose levels were affected by dopamine treatment were identified. By an in-depth analysis of the proteolytic peptides included in this list, eleven mitochondrial proteins showed altered proteolytic processing. One of these proteins (i.e., the 39S ribosomal protein L49 – MRPL49) was cleaved by the neprilysin protease, already exploited in clinics as a biotarget. We eventually demonstrated a mitochondrial subcellular localization of neprilysin in human cells for the first time. Collectively, these results shed new light on mitochondrial dysfunction linked to dopamine imbalance in PD and opened up the possibility to explore the mitochondrial targets of neprilysin as candidate biomarkers.

Molecular complexity of the major urinary protein system of the Norway rat, Rattus norvegicus

Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Cluster analysis shows the existence of only 10 predicted mup genes. Further, detailed sequencing of the urinary MUP isoforms reveals a less complex pattern of primary sequence polymorphism in the rat than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications, including the phosphorylation of Ser4 in some isoforms, and exoproteolytic trimming of specific isoforms. Our results raise the possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse. Shotgun proteomics data are available via ProteomExchange with identifier PXD013986.

Analysis of decolorization potential of Myrothecium roridum in the light of its secretome and toxicological studies

To identify the enzymes potentially useful for the decolorization of azo dyes, the secretome of the ascomycetous fungus Myrothecium roridum IM6482 was studied by using a bottom-up proteomic approach. Among the identified proteins, the most promising for dye removal was laccase, which decolorized respectively, 66, 91, 79, and 80% of Acid Blue 113 (AB 113), Acid Red 27 (AR 27), Direct Blue 14 (DB 14), and Acid Orange 7 (AO 7). The degradation of dyes was enhanced at the wide range of pH from 4 to 8. The addition of redox mediators allowed eliminating AB 113 in concentrations up to 400 mg/L and decolorization of the simulated textile effluent. Microbial toxicity and phytotoxicity tests indicated that dyes are converted into low-toxicity metabolites. This is the first insight into the M. roridum secretome, its identification and its application for removal of select azo dyes. Obtained results extended knowledge concerning biodegradative potential of ascomycetous, ligninolytic fungi and will contribute to the improvement of dye removal by fungi.

One-Pot Quantitative Top- and Middle-Down Analysis of GluC-Digested Histone H4.

Histone post-translational modifications (PTMs) have been intensively investigated due to their essential function in eukaryotic genome regulation. Histone modifications have been effectively studied using modified bottom-up proteomics approaches; however, the methods often do not capture single-molecule combinations of PTMs (proteoforms) that mediate known and expected biochemical mechanisms. Both middle-down mass spectrometry (MS) and top-down MS quantitation of H4 proteoforms present viable access to this important information. Histone H4 middle-down has previously avoided GluC digestion due to complex digestion products and interferences; however, the common AspN digestion cleaves at amino acid 23, disconnecting K31ac from other PTMs. Here, we demonstrate the effective use of GluC-based middle-down quantitation and compare it to top-down-based quantitation of proteoforms. Despite potential interferences in the m/z space, the proteoforms arising from all three GluC products (E52, E53, and E63) and intact H4 are chromatographically resolved and successfully analyzed in a single LC-MS analysis. Quantitative results and associated analytical metrics are compared between the different analytes of a single sample digested to different extents to reveal general concordance as well as the relative biases and complementarity of each approach. There is moderate proteoform discordance between digestion products (e.g., E52 and E53); however, each digestion product exhibits high concordance, regardless of digestion time. Under the conditions used, the GluC products are better chromatographically resolved yet show greater variance than the top-down quantitation that are more extensively sampled for MS2. GluC-based middle-down of H4 is thus viable. Both top-down and middle-down approaches have comparable quantitation capacity and are complementary.

Does oral fluid contribute to exhaled breath samples collected by means of an electret membrane?

To date, blood (and serum) as well as urine samples are the most commonly collected specimens for routine doping controls, which allow for the analytical coverage of an extensive set of target analytes relevant to sports drug testing programs. In the course of studies to identify potential alternative matrices to complement current testing approaches, exhaled breath (EB) has been found to offer advantageous properties especially with regard to the sample collection procedure, which is less invasive, less intrusive, and less time‐consuming when compared to conventional blood and urine testing. A yet unaddressed question has been the potential contribution of oral fluid (OF) to EB samples. The current investigation focused on characterizing an electret membrane‐based EB collection device concerning a potential introduction of OF during the sampling procedure. For that purpose, EB and OF samples collected under varying conditions from a total of 14 healthy volunteers were tested for the presence of abundant salivary proteins using bottom‐up proteomics approaches such as SDS‐PAGE followed by tryptic digestion and chromatographic‐mass spectrometric analysis. The trapping baffles integrated into the mouthpiece of the EB collection device were found to effectively retain OF introduced into the unit during sample collection as no saliva breakthrough was detectable using the established analytical approach targeting predominantly the highly abundant salivary α‐amylase. Since α‐amylase was found unaffected by storage, smoking, food intake, and exercise, it appears to be a useful marker to reveal possible OF contaminations of EB collection devices.

Coacervation of Lipid Bilayer in Natural Cell Membranes for Extraction, Fractionation, and Enrichment of Proteins in Proteomics Studies.

This is the first report where hexafluoroisopropanol (HFIP) was used to induce the coacervation of lipid components in natural cell membranes that would concomitantly result in solubilization, extraction, and enrichment of hydrophobic proteins (e.g., integral membrane proteins, IMP) into the coacervate phase, and extraction of hydrophilic proteins in a separate aqueous phase. The incorporation of this innovative approach in the proteomics workflow would allow the fractionation of proteins in separate aqueous and coacervate phases and would also eliminate the need for using surfactants. Subsequently, proteins can be identified by the bottom-up proteomics approach where samples were digested in solution after phase separation. Yeast cell wall proteins, anchored membrane proteins, and proteins related to some regulatory activities were mostly found in the aqueous-rich phase. On the other hand, most integral membrane proteins, proteins involved in metabolic processes, and proteins responsible for ions or drug binding were identified in the coacervate phase. The detergent-free, facile, and rapid process of natural lipid coacervation increased the number of identified proteins by 8% (vs no-phase separation experiment). The identification of all IMPs and organelle IMPs was improved by 13% and 29%, respectively. In addition, 25% more low-abundance proteins (less than 20 ppm) were identified.

Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey

We report on the variable venom composition of a population of the Caucasus viper (Vipera kaznakovi) in Northeastern Turkey. We applied a combination of venom gland transcriptomics, de-complexing bottom-up and top-down venomics. In contrast to sole bottom-up venomics approaches and gel or chromatography based venom comparison, our combined approach enables a faster and more detailed comparison of venom proteomes from multiple individuals. In total, we identified peptides and proteins from 15 toxin families, including snake venom metalloproteinases (svMP; 37.8%), phospholipases A2 (PLA2; 19.0%), snake venom serine proteinases (svSP; 11.5%), C-type lectins (CTL; 6.9%) and cysteine-rich secretory proteins (CRISP; 5.0%), in addition to several low abundant toxin families. Furthermore, we identified intraspecies variations of the venom composition of V. kaznakovi, and find these were mainly driven by the age of the animals, with lower svSP abundance detected in juveniles. On the proteoform level, several small molecular weight toxins between 5 and 8 kDa in size, as well as PLA2s, drove the differences observed between juvenile and adult individuals. This study provides novel insights into the venom variability of V. kaznakovi and highlights the utility of intact mass profiling for fast and detailed comparison of snake venom. Biological significancePopulation level and ontogenetic venom variation (e.g. diet, habitat, sex or age) can result in a loss of antivenom efficacy against snakebites from wide ranging snake populations. The current state of the art for the analysis of snake venoms are de-complexing bottom-up proteomics approaches. While useful, these have the significant drawback of being time-consuming and following costly protocols, and consequently are often applied to pooled venom samples. To overcome these shortcomings and to enable rapid and detailed profiling of large numbers of individual venom samples, we integrated an intact protein analysis workflow into a transcriptomics-guided bottom-up approach. The application of this workflow to snake individuals of a local population of V. kaznakovi revealed intraspecies variations in venom composition, which are primarily explained by the age of the animals, and highlighted svSP abundance to be one of the molecular drivers for the compositional differences observed.

Differential Urinary Proteomic Analysis of Endometrial Cancer.

Endometrial cancer is one of the most frequent gynecological malignancies present in more than 95 % of all uterine cancers. In spite of that, screening of such disease is not commonly performed in clinical practice due to enormous costs and relatively low sensitivity. Therefore, developing an effective screening test to diagnose endometrial cancer at early stages is of great importance for the clinical area of investigation. In this work, we applied urinary proteomics (i.e., bottom-up proteomic approach followed by nano HPLC-ESI-MS/MS) in patients with endometrial cancer, with respect to find proteins aimed for the early diagnostics and screening. According to the results, the significant semi-quantitative changes were observed in urinary proteome of treated patients. The proteins that may be pivotal in pathogenesis of endometrial cancer, like cadherin-1 (CDH1), vitronectin (VTN) and basement membrane specific-heparan sulphate proteoglycan core protein (HSPG2) were down-regulated, when compared to the control group. Ultimately, it can be stated that urinary proteomics has a potential for the searching of cancer protein biomarkers based on their altered concentration.

Products of Early and Advanced Glycation in the Soy Milk Proteome.

Thermal processing of soy milk kills pathogens and denatures anti-nutrition factors warranting microbiological safety, better digestibility, and longer storage. Additionally, Maillard reactions are triggered, yielding glycated proteins (Amadori/Heyns products) and a heterogeneous group of advanced glycation end-products (AGEs). These modifications alter the nutritional value, antigenicity, and digestibility of proteins. They also raise concerns about potentially toxic effects. This study aims at characterizing these modifications in proteins from different soy milk products. Here, glycation and AGE-modification sites in the proteome of ultrahigh-temperature-treated natural soy milk, soy milk sweetened with hexose (fructose)-containing sweeteners (SSM), and sucrose as well as soy-based infant formulas (SIFs) from different manufacturers are reported for the first time. A bottom-up proteomic approach based on nano reversed-phase high-perfomance liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoRP-HPLC-ESI-MS/MS) (collision-induced dissociation (CID) and electron transfer dissociation modes) identified 229 glycated peptides and 128 AGE-modified peptides resembling 53 proteins. The glycation sites are mainly derived from hexoses, whereas Nδ -carboxyethylarginine and methylglyoxal-derived hydroimidazolone are the main AGEs in soy milk. The qualitative and quantitative data obtained here indicate that early glycation increases with harsher processing conditions (SIFs) and the addition of hexose-containing sweeteners (SSMs), whereas the latter sweeteners (but not the harsher processing) triggered more AGE modifications.

Dynamin-Like Proteins of Endocytosis in Plants Are Coopted by Potyviruses To Enhance Virus Infection.

Endocytosis and endosomal trafficking regulate the proteins targeted to the plasma membrane and play essential roles in diverse cellular processes, including responses to pathogen attack. Here, we report the identification of Glycine max (soybean) endocytosis dynamin-like protein 5A (GmSDL5A) associated with purified soybean mosaic virus (SMV) virions from soybean using a bottom-up proteomics approach. Knockdown of GmSDL5A and its homologous gene GmSDL12A inhibits SMV infection in soybean. The role of analogous dynamin-like proteins in potyvirus infection was further confirmed and investigated using the Arabidopsis/turnip mosaic virus (TuMV) pathosystem. We demonstrate that dynamin-related proteins 2A and 2B in Arabidopsis thaliana (AtDRP2A, AtDRP2B), homologs of GmSDL5A, are recruited to the virus replication complex (VRC) of TuMV. TuMV infection is inhibited in both A. thalianadrp2a (atdrp2a) and atdrp2b knockout mutants. Overexpression of AtDRP2 promotes TuMV replication and intercellular movement. AtRDP2 interacts with TuMV VPg, CP, CI, and 6K2. Of these viral proteins, VPg, CP, and CI are essential for viral intercellular movement, and 6K2, VPg, and CI are critical components of the VRC. We reveal that VPg and CI are present in the punctate structures labeled by the endocytic tracer FM4-64, suggesting that VPg and CI can be endocytosed. Treatment of plant leaves with a dynamin-specific inhibitor disrupts the delivery of VPg and CI to endocytic structures and suppresses TuMV replication and intercellular movement. Taken together, these data suggest that dynamin-like proteins are novel host factors of potyviruses and that endocytic processes are involved in potyvirus infection.IMPORTANCE It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance, such as human and insect activities, to penetrate the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knockout of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases.

Snake venom proteome and immuno-profiling of the hundred-pace viper, Deinagkistrodon acutus, in Taiwan

Deinagkistrodon acutus, also known as the hundred-pace viper or Chinese moccasin, is a clinically significant venomous snake in Taiwan. To address the lack of knowledge on the venom proteome of D. acutus, the venom composition was studied by a bottom-up proteomic approach combining reverse phase high-performance liquid chromatography, SDS-PAGE, and LC–MS/MS analysis. The immunoreactivity and cross-reactivity of Taiwanese freeze-dried D. acutus antivenom (DA-AV) and hemorrhagic antivenom (FH-AV) were investigated, as well. The proteomic analysis revealed the presence of 29 distinct proteins from D. acutus venom belonging to 8 snake venom protein families. Snake venom metalloproteinase (SVMP, 46.86%), C-type lectin (CLEC, 37.59%), phospholipase A2 (PLA2, 7.33%) and snake venom serine protease (SVSP, 6.62%) were the most abundant proteins. In addition to DA-AV, FH-AV also showed a profile of broad immunorecognition toward the venom of D. acutus. Remarkably, both antivenoms specifically reacted with the HPLC fractions containing SVMPs, and the titer was 5–10 times higher than fractions of other components. This information helps us to deeply understand the pathophysiology of D. acutus envenomation and guide us to development of more effective antivenom for clinical treatment.