Neuroproteomics — LC-MS Quantitative Approaches

Neuroproteomics is a scientific field that aims to study all the proteins of the central nervous system, their expression, function, and interactions. The central nervous system is intricate and heterogeneous, and the study of its proteome is consequently complex, with many biological questions still requiring deep investigation. For this, mass spectrometry approaches, most often coupled with liquid chromatography (LC-MS), have been the number one choice in proteomics, and over the years it has added many important findings to the field. At this point it is important that proteomics turns to the quantitative expression of proteins instead of only identifying which proteins are present in a given sample, much because the most important alterations may be slight alterations in the quantity of a protein in a given situation. Therefore, many LC-MS quantitative approaches have been developed relying on the labeling of the proteins or even by using label-free techniques

Disclosing proteins in the leaves of cork oak plants associated with the immune response to Phytophthora cinnamomi inoculation in the roots: a long-term proteomics approach

The pathological interaction between oak trees and Phytophthora cinnamomi has implications in the cork oak decline observed over the last decades in the Iberian Peninsula. During host colonization, the phytopathogen secretes effector molecules like elicitins to increase disease effectiveness. The objective of this study was to unravel the proteome changes associated with the cork oak immune response triggered by P. cinnamomi inoculation in a long-term assay, through SWATH-MS quantitative proteomics performed in the oak leaves. Using the Arabidopis proteome database as a reference, 424 proteins were confidently quantified in cork oak leaves, of which 80 proteins showed a p-value below 0.05 or a fold-change greater than 2 or less than 0.5 in their levels between inoculated and control samples being considered as altered. The inoculation of cork oak roots with P. cinnamomi increased the levels of proteins associated with protein-DNA complex assembly, lipid oxidation, response to endoplasmic reticulum stress, and pyridine-containing compound metabolic process in the leaves. In opposition, several proteins associated with cellular metabolic compound salvage and monosaccharide catabolic process had significantly decreased abundances. The most significant abundance variations were observed for the Ribulose 1,5-Bisphosphate Carboxylase small subunit (RBCS1A), Heat Shock protein 90-1 (Hsp90-1), Lipoxygenase 2 (LOX2) and Histone superfamily protein H3.3 (A8MRLO/At4G40030) revealing a pertinent role for these proteins in the host-pathogen interaction mechanism. This work represents the first SWATH-MS analysis performed in cork oak plants inoculated with P. cinnamomi and highlights host proteins that have a relevant action in the homeostatic states that emerge from the interaction between the oomycete and the host in the long term and in a distal organ.FCT:UID/Multi/00631/2019 and UIDB/00631/2020 CEOT BASE to CEOT and ACC, GS and RP; UIDB/ 04326/2020 to CCMAR; Norma Transitoria- DL57/2016/CP1361/CT0015 to PP; contract NIBAP (ALG-01-0247-FEDER-037303) to RP; projects POCI-01-0145-FEDER-007440 (Ref. UIDB/04539/ 2020), POCI-01-0145-FEDER-016428 (Ref. SAICTPAC/0010/2015), POCI-01-0145- FEDER-029311 (Ref. PTDC/BTM-TEC/29311/ 2017), POCI-01-0145-FEDER-30943 (Ref. PTDC/ MECPSQ/30943/2017) and PTDC/MED-NEU/ 27946/2017 to CNC, BM and CS. The work at CNC was also funded by the National Mass Spectrometry Network (RNEM) under contract POCI-01-0145-FEDER-402-022125info:eu-repo/semantics/publishedVersio

Proteomics and antioxidant enzymes reveal different mechanisms of toxicity induced by ionic and nanoparticulate silver in bacteria

The increased use of silver nanoparticles (AgNPs) raises concerns about their impacts on aquatic ecosystems. The impacts of Ag+ and AgNPs were assessed on proteomic and antioxidant enzymatic responses of Pseudomonas sp. M1. The effects of Ag+ on bacterial growth were stronger than those of AgNPs (EC20 = 107.1 μg L−1 for Ag+; EC20 = 307.2 μg L−1 for AgNPs), indicating the lower toxicity of the latter. At EC20, the activities of antioxidant enzymes increased more under exposure to Ag+ than to AgNPs, particularly for superoxide dismutase and glutathione peroxidase (stimulation of 667% and 433%, respectively). A total of 166 proteins were identified by SWATH-MS; among these, only 59 had their content significantly altered by one or both forms of silver. Exposure to AgNPs resulted in an increase of about 54% of these proteins, whereas 54% decreased under exposure to Ag+. Gene Ontology enrichment analysis revealed that protein folding and transmembrane transport were the most relevant processes affected by Ag+ exposure, whereas AgNPs mostly affected translation. Also, results suggest that each form of silver induced different adaptive responses. Furthermore, the low levels of Ag+ released from AgNPs (<0.1%) support a minor role of dissolved silver in AgNP toxicity to Pseudomonas sp. M1ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by the Portuguese Foundation for Science and Technology I.P. (FCT) through the strategic funding UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), PTDC/AAC-AMB/121650/2010, POCI-01-0145-FEDER-007440 (ref. UID/NEU/04539/2013), the National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER402-022125 (ref. ROTEIRO/0028/2013), and PTDC/BIA-BMA/ 30922/201

Characterization of widespread proteome aggregation through aging in mammals

Proteome and proteostasis network disruptions accompany proteostasis alterations, leading to accumulation of protein aggregates, characteristic of several age-related diseases. Previous work in Caenorhabditis elegans and zebrafish unveiled asymptomatic protein aggregation (ARPA), characterized by generalized increased accumulation of insoluble proteins through aging. However, the consequences of protein homeostasis imbalances in the context of healthy aging in mammals remains mostly unexplored. To elucidate if accumulation of insoluble proteins also occurs through aging in mammals, C57BL/6 mice with different ages corresponding to young (6 months old), adult (13 months old), and old stages (18 and 24 months old) were used. Detergent-insoluble fractions were isolated from total protein extracts of tissues, followed by characterization of both total and detergent-insoluble protein profiles. Our results show tissue-specific proteome alterations through aging. For example, the insoluble fractions increase through aging in brain-derived tissues, but muscular tissues do not display significant alterations until 18 months old. We are now performing SWATH-MS analysis to identify differential proteome signatures of aging and which proteins are more prone to aggregate to further elucidate the functions and biological processes affected by ARPA.publishe

Proteome dataset of sea bass (Dicentrarchus labrax) skin-scales exposed to fluoxetine and estradiol

Contamination of aquatic ecosystems with anthropogenic pollutants, including pharmaceutical drugs, is a major concern worldwide. Aquatic organisms such as fish are particularly at risk of exposure to pollutants. The surface of fish is the first point of contact with pollutants, but few studies have considered the impact of pollutants on the skin-scale barrier. The present proteome data are the basis of the findings discussed in the associated research article "Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol" [1]. Juvenile sea bass were exposed by intraperitoneal injections to: a) the antidepressant fluoxetine (FLX), a widely prescribed psychotropic drug and an emerging pollutant; b) the natural estrogen 17 beta-estradiol (E2) and c) the vehicle, coconut oil (control). The scale proteome of fish exposed to these compounds for 5 days was analysed using quantitative label-free proteomics technology SWATH-MS (sequential windowed data-independent a cquisition of the total high-resolution-mass spectra). The proteome data generated was submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020983. LC-MS data from pooled protein extracts from the scales of all experimental groups was acquired using information-dependent acquisition (IDA) and 1,254 proteins were identified by searching against the sea bass genome database. 715 proteins were quantified by SWATH acquisition, and 213 proteins had modified levels (p < 0.05) between the E2- or FLX-exposed fish compared to the control. The main biological processes and KEGG pathways affected by E2 or FLX treatments were identified using Cytoscape/ClueGO enrichment analyses. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)info:eu-repo/semantics/publishedVersio

Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology

Background: Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells. Methods: The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses. Results: We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. Conclusions: These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.The authors would like to acknowledge the funding agencies that supported this work: Fundacao para a Ciencia e Tecnologia (FCT), Portugal, projects reference: PTDC/SAU-GMG/113795/2009 (BMC); SFRH/BD/88121/2012 (JVdC); SFRH/BD/103075/2014 (EDG); IF/00601/2012 (BMC); IF/00111/2013 (AJS); SFRH/BD/81495/2011 (SIA); PTDC/NEU-NMC/0205/2012, PTDC/NEUSCC/ 7051/2014, PEst-C/SAU/LA0001/2013-2014 and UID/NEU/04539/2013 (BM); Fundacao Calouste Gulbenkian (BMC); Liga Portuguesa Contra o Cancro (BMC); " COMPETE Programa Operacional Factores de Competitividade, QREN, the European Union (FEDER-Fundo Europeu de Desenvolvimento Regional) and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005; FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038; and project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funding body did not have a role in the design of the study, in collection, analysis or interpretation of data, or in writing the manuscript

COVID-19 salivary protein profile: unravelling molecular aspects of SARS-CoV-2 infection

COVID-19 is the most impacting global pandemic of all time, with over 600 million infected and 6.5 million deaths worldwide, in addition to an unprecedented economic impact. Despite the many advances in scientific knowledge about the disease, much remains to be clarified about the molecular alterations induced by SARS-CoV-2 infection. In this work, we present a hybrid proteomics and in silico interactomics strategy to establish a COVID-19 salivary protein profile. Data are available via ProteomeXchange with identifier PXD036571. The differential proteome was narrowed down by the Partial Least-Squares Discriminant Analysis and enrichment analysis was performed with FunRich. In parallel, OralInt was used to determine interspecies Protein-Protein Interactions between humans and SARS-CoV-2. Five dysregulated biological processes were identified in the COVID-19 proteome profile: Apoptosis, Energy Pathways, Immune Response, Protein Metabolism and Transport. We identified 10 proteins (KLK 11, IMPA2, ANXA7, PLP2, IGLV2-11, IGHV3-43D, IGKV2-24, TMEM165, VSIG10 and PHB2) that had never been associated with SARS-CoV-2 infection, representing new evidence of the impact of COVID-19. Interactomics analysis showed viral influence on the host immune response, mainly through interaction with the degranulation of neutrophils. The virus alters the host’s energy metabolism and interferes with apoptosis mechanisms.info:eu-repo/semantics/publishedVersio

Specific Antiproliferative Properties of Proteinaceous Toxin Secretions from the Marine Annelid Eulalia sp. onto Ovarian Cancer Cells

The Portuguese Foundation for Science and Technology (FCT) funded project WormALL (PTDC/BTA-BTA/28650/2017) plus the grants SFRH/BD/109462/2015 to A.P.R., and CEECIND/02699/2017 to A.R.G. The Applied Molecular Biosciences Unit-UCIBIO is financed by national funds from FCT, ref. UIDB/04378/2020. FCT, along with the European Regional Development Fund (ERDF) through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation also funded the projects: POCI-01-0145-FEDER-007440 (UID/NEU/04539/2019), POCI-01-0145-FEDER-016428 (SAICTPAC/0010/2015), POCI-01-0145-FEDER-029311 (PTDC/BTM-TEC/29311/2017), POCI-01-0145-FEDER-30943 (PTDC/MEC-PSQ/30943/2017) and PTDC/MED-NEU/27946/2017. The work was also funded by the National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER-402-022125 (ref.: ROTEIRO/0028/2013).As Yondelis joins the ranks of approved anti-cancer drugs, the benefit from exploring the oceans' biodiversity becomes clear. From marine toxins, relevant bioproducts can be obtained due to their potential to interfere with specific pathways. We explored the cytotoxicity of toxin-bearing secretions of the polychaete Eulalia onto a battery of normal and cancer human cell lines and discovered that the cocktail of proteins is more toxic towards an ovarian cancer cell line (A2780). The secretions' main proteins were identified by proteomics and transcriptomics: 14-3-3 protein, Hsp70, Rab3, Arylsulfatase B and serine protease, the latter two being known toxins. This mixture of toxins induces cell-cycle arrest at G2/M phase after 3h exposure in A2780 cells and extrinsic programmed cell death. These findings indicate that partial re-activation of the G2/M checkpoint, which is inactivated in many cancer cells, can be partly reversed by the toxic mixture. Protein-protein interaction networks partake in two cytotoxic effects: cell-cycle arrest with a link to RAB3C and RAF1; and lytic activity of arylsulfatases. The discovery of both mechanisms indicates that venomous mixtures may affect proliferating cells in a specific manner, highlighting the cocktails' potential in the fine-tuning of anti-cancer therapeutics targeting cell cycle and protein homeostasis.publishersversionpublishe

Self-recycling and partially conservative replication of mycobacterial methylmannose polysaccharides

The steep increase in nontuberculous mycobacteria (NTM) infections makes understanding their unique physiology an urgent health priority. NTM synthesize two polysaccharides proposed to modulate fatty acid metabolism: the ubiquitous 6-O-methylglucose lipopolysaccharide, and the 3-O-methylmannose polysaccharide (MMP) so far detected in rapidly growing mycobacteria. The recent identification of a unique MMP methyltransferase implicated the adjacent genes in MMP biosynthesis. We report a wide distribution of this gene cluster in NTM, including slowly growing mycobacteria such as Mycobacterium avium, which we reveal to produce MMP. Using a combination of MMP purification and chemoenzymatic syntheses of intermediates, we identified the biosynthetic mechanism of MMP, relying on two enzymes that we characterized biochemically and structurally: a previously undescribed ?-endomannosidase that hydrolyses MMP into defined-sized mannoligosaccharides that prime the elongation of new daughter MMP chains by a rare ?-(1?4)-mannosyltransferase. Therefore, MMP biogenesis occurs through a partially conservative replication mechanism, whose disruption affected mycobacterial growth rate at low temperature

Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue-Derived Stem Cells, and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis

The use of human mesenchymal stem cells (hMSCs) has emerged as a possible therapeutic strategy for CNS-related conditions. Research in the last decade strongly suggests that MSC-mediated benefits are closely related with their secretome. Studies published in recent years have shown that the secretome of hMSCs isolated from different tissue sources may present significant variation. With this in mind, the present work performed a comparative proteomic-based analysis through mass spectrometry on the secretome of hMSCs derived from bone marrow (BMSCs), adipose tissue (ASCs), and human umbilical cord perivascular cells (HUCPVCs). The results revealed that BMSCs, ASCs, and HUCPVCs differed in their secretion of neurotrophic, neurogenic, axon guidance, axon growth, and neurodifferentiative proteins, as well as proteins with neuroprotective actions against oxidative stress, apoptosis, and excitotoxicity, which have been shown to be involved in several CNS disorder/injury processes. Although important changes were observed within the secretome of the cell populations that were analyzed, all cell populations shared the capability of secreting important neuroregulatory molecules. The difference in their secretion pattern may indicate that their secretome is specific to a condition of the CNS. Nevertheless, the confirmation that the secretome of MSCs isolated from different tissue sources is rich in neuroregulatory molecules represents an important asset not only for the development of future neuroregenerative strategies but also for their use as a therapeutic option for human clinical trials.Foundation Calouste de Gulbenkian for the funds attributed to A.J.S.; Portuguese Foundation for Science and Technology (FCT) PhD fel- lowships attributed to A.O.P. (SFRH/BD/33900/2009) and S.I.A. (SFRH/BD/81495/2011) and Ciência 2007, IF Development Grant attributed to A.J.S., and projects PTDC/ NEU-NMC/0205/2012, UID/NEU/04539/2013; cofinanced by COMPETE Programa Operacional Factores de Compe- titividade; and by The National Mass Spectrometry Network (RNEM) (REDE/1506/REM/2005); Prémios Santa Casa Neurociências—Prize Melo e Castro for Spinal Cord Injury Research; cofunded by Programa Operacional Regional do Norte (ON.2–O Novo Norte),ao abrigo do Quadro de Referência Estratégico Nacional (QREN), and através do Fundo Europeu de Desenvolvimento Regional (FEDER). The authors also would like to thank Professor J.E.D. (University of Toronto, Canada) and Professor J.M.G. (Tulane University) for kindly providing HUCPVCs and ASCs, respectivelyinfo:eu-repo/semantics/publishedVersio