Proteomic Profiling of Lymph Nodes Differentiates Classic Hodgkin Lymphoma With and Without Skeletal Involvement.

Classic Hodgkin lymphoma (CHL) is a highly curable disease, even in advanced stages. Controversy remains over whether bone involvement negatively affects overall and progression-free survival in patients treated with intensive chemotherapy regimens. Whether cases that present with bone lesions harbor specific tumor microenvironmental features is unknown. We investigated protein expression in diagnostic lymph node biopsies from CHL patients with and without skeletal involvement at diagnosis to identify potential markers of skeletal disease. Protein expression patterns in diagnostic formalin-fixed paraffin-embedded lymphoma lymph node samples from CHL patients were analyzed by nano-liquid chromatography-tandem mass spectrometry. Patients were grouped according to skeletal involvement, which was defined as the presence of one or more FDG-avid lesions on a diagnostic FDG-PET/CT scan. Protein profiles identified patients with skeletal disease at diagnosis and showed disrupted cellular pathways, including immune system processes, cell adhesion, and cell growth/survival. Immunohistochemical evaluation also demonstrated differential expressions of angiotensin-converting enzyme (ACE), intercellular adhesion molecule 3 (ICAM3), integrin alpha-X (ITGAX), and calreticulin (CALR). In conclusion, proteomics identified altered protein expression profiles in lymph nodes among CHL cases presenting with disease disseminated to the skeletal system, which implies altered disease pathogenesis for these patients.

Fibrogenic signals persist in DAA-treated HCV patients after sustained virological response.

BACKGROUND & AIMS: Patients with HCV who achieve a sustained virological response (SVR) on direct-acting antiviral (DAA) therapy still need to be monitored for signs of liver disease progression. To this end, the identification of both disease biomarkers and therapeutic targets is necessary. METHODS: Extracellular vesicles (EVs) purified from plasma of 15 healthy donors (HDs), and 16 HCV-infected patients before (T0) and after (T6) DAA treatment were utilized for functional and miRNA cargo analysis. EVs purified from plasma of 17 HDs and 23 HCV-infected patients (T0 and T6) were employed for proteomic and western blot analyses. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry and western blot. RESULTS: On the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HDs, T0 and T6, we performed structural analysis of EVs. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122-5p were statistically underrepresented in T0 EVs compared to HD EVs, while miR204-5p and miR143-3p were statistically underrepresented in T6 EVs compared to HD EVs (p <0.05); (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HDs; among them, the fibrogenic protein DIAPH1 was upregulated (Log2 fold change of 4.4). CONCLUSIONS: Taken together, these results highlight structural EV modifications that are conceivably causal for long-term liver disease progression in patients with HCV despite DAA-mediated SVR. LAY SUMMARY: Direct-acting antivirals lead to virological cure in the majority of patients with chronic hepatitis C virus infection. However, the risk of liver disease progression or complications in patients with fibrosis and cirrhosis remains in some patients even after virological cure. Herein, we show that extracellular vesicle modifications could be linked to long-term liver disease progression in patients who have achieved virological cure; these modifications could potentially be used as biomarkers or treatment targets in such patients.

Search for Novel Plasma Membrane Proteins as Potential Biomarkers in Human Mesenchymal Stem Cells Derived from Dental Pulp, Adipose Tissue, Bone Marrow, and Hair Follicle.

One of the drawbacks preventing the use of mesenchymal stem cells (MSCs) in clinical practice is the heterogeneous nature of their cultures. MSC cultures are not homogeneously formed by the MSCs and may contain non-mesenchymal cell types. Therefore, prior to use in clinics or research, complete characterization of MSCs should be performed to demonstrate the existence or absence of proper stem cell markers, many of which are happened to be cell-surface proteins. Unfortunately, the success of MSC characterization studies is limited due to the low specificity of the currently available cell-surface markers. Therefore, in this study, we aimed to investigate the plasma membrane (PM) proteins of MSCs isolated from human dental pulp (DP), adipose tissue (AT), bone marrow (BM), and hair follicle (HF) with the hope of proposing novel putative specific MSC markers. Differential-velocity centrifugation was used to enrich PM proteins. The isolated proteins were then identified by nLC-MS/MS and subjected to bioinformatics analysis. Proteins that were unique to each MSC type (CD9, CD10, CD63 for DP-MSCs; CD26, CD81, CD201, CD364 for AT-MSCs; Cd49a, CD49d for HF-MSCs; CD49e, CD56, CD92, CD97, CD156b, CD156c, CD220, CD221, CD298, CD315 for BM-MSCs) and common to all four MSC types (CD13, CD29, CD44, CD51, CD59, CD73, CD90) were identified. Uncharacterized proteins that have transmembrane (TM) domains were also detected. Some of the proteins identified in this study were the putative cell-surface markers that might be used for characterization of MSCs.

Quantitative evaluation of liposomal doxorubicin and its metabolites in spheroids.

Accurate measurement and understanding of therapeutic uptake and metabolism is key in the drug development process. This work examines the amount of doxorubicin that can penetrate into spheroids after being encapsulated in a liposomal configuration in comparison with free drug. Through a process known as serial trypsinization, three distinct cellular populations of a spheroid were successfully separated and a small molecule extraction was used to isolate the chemotherapeutic. Doxorubicin showed a time-dependent permeability into spheroids with the most drug accumulating in the core at 24 h of treatment. Entrapment of the chemotherapeutic delayed the permeability of the drug and resulted in reduced amounts quantified at the earlier time points. These findings validate the claim that liposomal therapeutics have the ability to alter the pharmacokinetics and pharmacodynamics profiles of a drug while also demonstrating the combined power of mass spectrometry and three-dimensional cell cultures to evaluate drug penetration and metabolism. Graphical abstract.

A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds.

Earlier this year we published a method article aimed at optimising protein extraction from mature buds of medicinal cannabis for trypsin-based shotgun proteomics (Vincent, D., et al. Molecules 2019, 24, 659). We then developed a top-down proteomics (TDP) method (Vincent, D., et al. Proteomes 2019, 7, 33). This follow-up study aims at optimising the digestion of medicinal cannabis proteins for identification purposes by bottom-up and middle-down proteomics (BUP and MDP). Four proteases, namely a mixture of trypsin/LysC, GluC, and chymotrypsin, which target different amino acids (AAs) and therefore are orthogonal and cleave proteins more or less frequently, were tested both on their own as well as sequentially or pooled, followed by nLC-MS/MS analyses of the peptide digests. Bovine serum albumin (BSA, 66 kDa) was used as a control of digestion efficiency. With this multiple protease strategy, BSA was reproducibly 97% sequenced, with peptides ranging from 0.7 to 6.4 kD containing 5 to 54 AA residues with 0 to 6 miscleavages. The proteome of mature apical buds from medicinal cannabis was explored more in depth with the identification of 27,123 peptides matching 494 unique accessions corresponding to 229 unique proteins from Cannabis sativa and close relatives, including 130 (57%) additional annotations when the list is compared to that of our previous BUP study (Vincent, D., et al. Molecules 2019, 24, 659). Almost half of the medicinal cannabis proteins were identified with 100% sequence coverage, with peptides composed of 7 to 91 AA residues with up to 9 miscleavages and ranging from 0.6 to 10 kDa, thus falling into the MDP domain. Many post-translational modifications (PTMs) were identified, such as oxidation, phosphorylations, and N-terminus acetylations. This method will pave the way for deeper proteome exploration of the reproductive organs of medicinal cannabis, and therefore for molecular phenotyping within breeding programs.

Optimisation of Protein Extraction from Medicinal Cannabis Mature Buds for Bottom-Up Proteomics.

Medicinal cannabis is used to relieve the symptoms of certain medical conditions, such as epilepsy. Cannabis is a controlled substance and until recently was illegal in many jurisdictions. Consequently, the study of this plant has been restricted. Proteomics studies on Cannabis sativa reported so far have been primarily based on plant organs and tissues other than buds, such as roots, hypocotyl, leaves, hempseeds and flour. As far as we know, no optimisation of protein extraction from cannabis reproductive tissues has been attempted. Therefore, we set out to assess different protein extraction methods followed by mass spectrometry-based proteomics to recover, separate and identify the proteins of the reproductive organs of medicinal cannabis, apical buds and isolated trichomes. Database search following shotgun proteomics was limited to protein sequences from C. sativa and closely related species available from UniprotKB. Our results demonstrate that a buffer containing the chaotrope reagent guanidine hydrochloride recovers many more proteins than a urea-based buffer. In combination with a precipitation with trichloroacetic acid, such buffer proved optimum to identify proteins using a trypsin digestion followed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analyses. This is validated by focusing on enzymes involved in the phytocannabinoid pathway.

Corrigendum: Integrated metabolomics and proteomics reveal biomarkers associated with hemodialysis in end-stage kidney disease.

[This corrects the article DOI: 10.3389/fphar.2023.1243505.].

Integrated metabolomics and proteomics reveal biomarkers associated with hemodialysis in end-stage kidney disease.

Background: We hypothesize that the poor survival outcomes of end-stage kidney disease (ESKD) patients undergoing hemodialysis are associated with a low filtering efficiency and selectivity. The current gold standard criteria using single or several markers show an inability to predict or disclose the treatment effect and disease progression accurately. Methods: We performed an integrated mass spectrometry-based metabolomic and proteomic workflow capable of detecting and quantifying circulating small molecules and proteins in the serum of ESKD patients. Markers linked to cardiovascular disease (CVD) were validated on human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Results: We identified dozens of elevated molecules in the serum of patients compared with healthy controls. Surprisingly, many metabolites, including lipids, remained at an elevated blood concentration despite dialysis. These molecules and their associated physical interaction networks are correlated with clinical complications in chronic kidney disease. This study confirmed two uremic toxins associated with CVD, a major risk for patients with ESKD. Conclusion: The retained molecules and metabolite-protein interaction network address a knowledge gap of candidate uremic toxins associated with clinical complications in patients undergoing dialysis, providing mechanistic insights and potential drug discovery strategies for ESKD.

Proteomic analysis of the mouse sperm acrosome - towards an understanding of an organelle with diverse functionality.

The acrosome located within the mammalian sperm head is essential for successful fertilization, as it enables the sperm to penetrate the extracellular layers of the oocyte and fuse with oolemma. However, the mammalian acrosomal vesicle is no longer considered to contain only hydrolytic enzymes. Using label-free nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics, we identified a total of 885 proteins in the acrosome isolated from spermatozoa obtained from cauda epididymis of free-living house mice Mus musculus musculus contains a total of 885 proteins. Among these, 334 proteins were significantly enriched in the acrosome thus representing 27.3% of the whole proteome of the intact sperm. Importantly, we have detected a total of nine calycins while eight of them belong to the lipocalin protein family. In mice, lipocalins are involved in multi-level chemical communication between individuals including pheromone transport and odor perception. Using an indirect immunofluorescence assay, we demonstrated that lipocalin 5 (LCN5) is expressed in the mouse germ cells, and after completing spermatogenesis, it remains localized in the sperm acrosome until the last step of the extratesticular maturation, the acrosome reaction. The presence of lipocalins in the acrosome and acrosome-reacted sperm suggests their original role as chelators of organic and potentially toxic compounds resulting from ongoing spermiogenesis. Along with this evidence, detected mitochondrial (e.g., a subunit of the cytochrome c oxidase MTCO1) and proteasomal proteins (subunits of both 20 S core proteasome [PSMA2, PSMBs] and 19 S regulatory particle [PSMDs]) in acrosomes provide further evidence that acrosomes could also function as `waste baskets` after testicular sperm maturation.

Label-free nLC-MS/MS proteomic analysis reveals significant differences in the proteome between colorectal cancer tissues and normal colon mucosa.

Despite the discovery of numerous driving and passenger genes that play key roles in cancer characteristics, progress in cancer treatment has not been satisfactory. This is mainly because conventional therapies are neither selective nor targeted. Another important reason is that cancer cells rapidly develop resistance to chemotherapeutic agents due to excessive accumulation of mutations and/or epigenetic changes. In light of this, we believe that the discovery of new targets and key genes/proteins could improve treatment options. In this study, tissue samples (tumor and normal mucosa) were first collected from the colon or rectum by right or left hemicolectomy. Proteomic analysis was then performed using the label-free nLC-MS/MS method. We determined 77 proteins with statistically significant differences in expression levels between cancerous and normal mucosa. While the expression of 76 proteins was decreased in cancer tissues, only one protein (RNA-binding motif protein_X chromosome-RBMX) was increased in colorectal cancer tissues. The bioinformatics portal Metascape was used to determine the biological processes involved. 77 proteins with significantly different expression between cancerous and normal tissues were compared with the UALCAN platform using data from the Clinical Proteomics Tumor Analysis Consortium (CPTAC). The results for 45 of the 77 proteins clearly matched the CPTAC dataset. Western blot studies confirmed that RBMX protein (critical for gene transcription and alternative splicing of various pre-mRNAs) was increased 2.04-fold, while decorin protein (a matrix proteoglycan with tumor suppressor functions) was dramatically decreased by about 6.04-fold in tumor samples compared with normal mucosa.

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis.

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed. Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1'681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Amaranth calcium oxalate crystals are associated with chloroplast structures and proteins.

Calcium oxalate (CaOx) crystals in plants are formed in crystal idioblasts cells and have specific geometric shapes. Their proposed functions include calcium homeostasis and carbon source, among others. Amaranth is a plant that presents high tolerance to abiotic stresses and accumulates considerable amounts of CaOx crystals; however, few studies have focused on characterizing the crystals ultrastructure and none is related to identifying proteins bound to them. This information is of great interest to understand the mechanisms related to CaOx crystal formation and to support their proposed functions. Thus, this work aimed to characterize CaOx crystals in amaranth leaves. Crystals were purified and the proteins bound to them were isolated and identified by nLC-MS/MS. Leaf sections were analyzed by light and electron microscopy. The identified proteins were related to the chloroplast such as ATPb synthase, RuBisCO large subunit, and cell wall-related proteins, which were validated by immunohistochemistry and immunogold labeling. In addition, it was observed that CaOx crystal idioblasts were formed from parenchyma cells associated with mesophyll and veins, in which the thylakoid membranes of degraded chloroplasts turned into crystal chambers. These results significantly advance our understanding of the mechanisms of CaOx crystal formation and the potential function as an alternative carbon source in leaves.

Impact of different oral treatments on the composition of the supragingival plaque microbiome.

Background: Dental plaque consists of a diverse microbial community embedded in a complex structure of exopolysaccharides. Dental biofilms form a natural barrier against pathogens but lead to oral diseases in a dysbiotic state. Objective: Using a metaproteome approach combined with a standard plaque-regrowth study, this pilot study examined the impact of different concentrations of lactoperoxidase (LPO) on early plaque formation, and active biological processes. Design: Sixteen orally healthy subjects received four local treatments as a randomized single-blind study based on a cross-over design. Two lozenges containing components of the LPO-system in different concentrations were compared to a placebo and Listerine®. The newly formed dental plaque was analyzed by mass spectrometry (nLC-MS/MS). Results: On average 1,916 metaproteins per sample were identified, which could be assigned to 116 genera and 1,316 protein functions. Listerine® reduced the number of metaproteins and their relative abundance, confirming the plaque inhibiting effect. The LPO-lozenges triggered mainly higher metaprotein abundances of early and secondary colonizers as well as bacteria associated with dental health but also periodontitis. Functional information indicated plaque biofilm growth. Conclusion: In conclusion, the mechanisms on plaque biofilm formation of Listerine® and the LPO-system containing lozenges are different. In contrast to Listerine®, the lozenges led to a higher bacterial diversity.

Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.

Identification of Potential Peptide Inhibitors of ACE-2 Target of SARS-CoV-2 from Buckwheat & Quinoa.

It is well established fact that peptides from various foods offer human health benefits displaying diverse functionalities. Millets considered as super foods is a major alternative in recent days for traditional diet being rich in proteins and fibre along with trace minerals and vitamins. In this connection, proteins from Buckwheat and Quinoa were digested by in vitro simulation digestion for the generation of peptides, analyzed by nLC-MS/MS and the functional annotations of the identified proteins/peptides were carried out. The study led to the identification of 34 small peptides and their parent proteins clustered into 4 gene functional groups and their localization prediction indicated their involvement in energy metabolism, transport and storage. Interestingly, the identified peptides maximally displayed DPP-IV and ACE inhibitions. The present study was extended to unravel ACE-2 inhibition targeting COVID-19 by selecting ACE-2-Spike binding domain for molecular docking studies. The NWRTVKYG interacted with the ACE-2-Spike interface displaying the feasible binding energy (- 213.63) and docking score (- 12.43) and the MD simulation revealed the ability of the peptide in stabilizing the protein-peptide composite. The present investigation thus establishes newer vista for food derived peptides having ACE-2 inhibitory potential as tentative strategy for SARS-CoV-2 therapeutics.

Bottom-Up Community Proteome Analysis of Saliva Samples and Tongue Swabs by Data-Dependent Acquisition Nano LC-MS/MS Mass Spectrometry.

Analysis using mass spectrometry enables the characterization of metaproteomes in their native environments and overcomes the limitation of proteomics of pure cultures. Metaproteomics is a promising approach to link functions of currently actively expressed genes to the phylogenetic composition of the microbiome in their habitat. In this chapter, we describe the preparation of saliva samples and tongue swabs for nLC-MS/MS measurements and their bioinformatic analysis based on the Trans-Proteomic Pipeline and Prophane to study the oral microbiome .

Alterations in the Proteome and Phosphoproteome Profiles of Rat Hippocampus after Six Months of Morphine Withdrawal: Comparison with the Forebrain Cortex.

The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

Impact of three-month morphine withdrawal on rat brain cortex, hippocampus, striatum and cerebellum: proteomic and phosphoproteomic studies.

Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes. The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus. Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.

Functional milk proteome analysis of genetically diverse goats from different agro climatic regions.

Goat milk, a choice of substitution to mother's milk for its composition, fulfils nutritional requirement of infants, pregnant mothers and older people. The present study was carried out to unravel the milk proteome profiles from geographically and genetically diverse goat breeds by gel based 2DE and nLC-MS/MS. A total of 1307 functional proteins comprising casein and other low abundance proteins were identified. Gene annotations revealed that the majority of the proteins were involved in binding function, catalytic activity and structural molecules and localised in nucleus and membrane. The distinguished proteins were involved in 144 KEGG pathways in information processing, metabolism, cellular process, organismal systems and diseases. The large number of proteins and peptides including bioactive peptides were reported from goat milk from diverse agro-climatic regions of India indicating their significant potential for human health applications. SIGNIFICANCE: Goat milk in India is used in various Ayurvedic formulations to treat a number of ailments and allergies as well as for nutraceutical formulations. The study identifies milk protein variants both at protein and DNA level and subsequent identification of proteins by 2DE and nLC-MS/MS resulting in a proteome comprising of 1307 proteins. The specific proteins and peptides having significant role in immune regulation, disease pathways, cellular growth and metabolism have been identified. The results contribute to goat milk protein and peptide database which is very limited. We identified proteins for specific functional categories and associated them with different pathways for studying functional diversity of goat milk proteins. The proteins and peptides identified can be used for multiple human health application.

Dual Element (C/Cl) Isotope Analysis Indicates Distinct Mechanisms of Reductive Dehalogenation of Chlorinated Ethenes and Dichloroethane in Dehalococcoides mccartyi Strain BTF08 With Defined Reductive Dehalogenase Inventories.

Dehalococcoides mccartyi strain BTF08 has the unique property to couple complete dechlorination of tetrachloroethene and 1,2-dichloroethane to ethene with growth by using the halogenated compounds as terminal electron acceptor. The genome of strain BTF08 encodes 20 genes for reductive dehalogenase homologous proteins (RdhA) including those described for dehalogenation of tetrachloroethene (PceA, PteA), trichloroethene (TceA) and vinyl chloride (VcrA). Thus far it is unknown under which conditions the different RdhAs are expressed, what their substrate specificity is and if different reaction mechanisms are employed. Here we found by proteomic analysis from differentially activated batches that PteA and VcrA were expressed during dechlorination of tetrachloroethene to ethene, while TceA was expressed during 1,2-dichloroethane dehalogenation. Carbon and chlorine compound-specific stable isotope analysis suggested distinct reaction mechanisms for the dechlorination of (i) cis-dichloroethene and vinyl chloride versus (ii) tetrachloroethene. This differentiation was observed independent of the expressed RdhA proteins. Differently, two stable isotope fractionation patterns were observed for 1,2-dichloroethane transformation, for cells with distinct RdhA inventories. Conclusively, we could link specific RdhA expression with functions and provide an insight into the apparently substrate-specific reaction mechanisms in the pathway of reductive dehalogenation in D. mccartyi strain BTF08. Data are available via ProteomeXchange with identifiers PXD018558 and PXD018595.