Isolation of Extracellular Vesicles Using Titanium Dioxide Microspheres.

Extracellular vesicles (EVs) are bilayer membrane particles released from several cell types to the extracellular environment. EVs have a crucial role in cell-cell communication, involving different biological processes in health and diseases. Due to the potential of biomarkers for several diseases as diagnostic and therapeutic tools, it is relevant to understand the biology of the EVs and their content. One of the current challenges involving EVs is regarding the purification method, which is a critical step for EV's functional and characterization studies. Ultracentrifugation is the most used method for EV isolation, where the nanoparticles are separated in sequential centrifugation to isolate the EVs based on their size. However, for viscous biofluids such as plasma, there is a co-isolation of the most abundant proteins, which can impair the EV's protein identification due to the low abundance of these proteins and signal suppression by the most abundant plasma proteins. Emerging techniques have gained attention in recent years. Titanium dioxide (TiO2) is one of the most promising techniques due to its property for selective isolation based on the interaction with phospholipids in the EV membrane. Using a small amount of TiO2 beads and a low volume of plasma, it is possible to isolate EVs with reduced plasma protein co-isolation. This study describes a comprehensive workflow for the isolation and characterization of plasma extracellular vesicles (EVs) using mass spectrometry-based proteomics techniques. The aim of this chapter is describe the EV isolation using TiO2 beads enrichment and high-throughput mass spectrometry techniques to efficiently identify the protein composition of EVs in a fast and straightforward manner.

Mass Spectrometry-Based Characterization of Protein Aggregates in Tissues and Biofluids.

Protein aggregation is a common mechanism in multiple neurodegenerative and heart diseases and the accumulation of proteins in aggregates is toxic to cells, causing injury and death. The degree of protein aggregation directly correlates with the severity of the disease. Misfolded proteins present thermodynamic barriers that culminate in the loss of structure and function and the exposure of hydrophobic residues. The exposure of hydrophobic residues is the driving force behind protein aggregation, as it reduces surface free energy and increases the propensity for the formation of large insoluble aggregates. Exploring the protein content of aggregates is fundamental to understanding their formation mechanism and pathophysiological effects. We demonstrate here a method for isolating aggregated protein content in human plasma and mouse brain samples. The samples were characterized by mass spectrometry analysis, transmission electron microscopy, and western blotting. We report the identification of proteins associated with neurodegenerative diseases in the isolated pellets. The western blotting analyses of the isolated pellet showed the positivity for CD89 and CD63, consolidated markers of exosomes, confirming the presence of exosomes within the pellet but not in the supernatant in human plasma. Notably, the concomitant isolation of exosomes together with the protein aggregates was feasible starting from 200 µL of human plasma. Moreover, the presented methodology separated albumin from the aggregated pellet, allowing identification of larger diversity of proteins through mass spectrometry analysis.

A computational pipeline elucidating functions of conserved hypothetical Trypanosoma cruzi proteins based on public proteomic data.

The proteome is complex, dynamic, and functionally diverse. Functional proteomics aims to characterize the functions of proteins in biological systems. However, there is a delay in annotating the function of proteins, even in model organisms. This gap is even greater in other organisms, including Trypanosoma cruzi, the causative agent of the parasitic, systemic, and sometimes fatal disease called Chagas disease. About 99.8% of Trypanosoma cruzi proteome is not manually annotated (unreviewed), among which>25% are conserved hypothetical proteins (CHPs), calling attention to the knowledge gap on the protein content of this organism. CHPs are conserved proteins among different species of various evolutionary lineages; however, they lack functional validation. This study describes a bioinformatics pipeline applied to public proteomic data to infer possible biological functions of conserved hypothetical Trypanosoma cruzi proteins. Here, the adopted strategy consisted of collecting differentially expressed proteins between the epimastigote and metacyclic trypomastigotes stages of Trypanosoma cruzi; followed by the functional characterization of these CHPs applying a manifold learning technique for dimension reduction and 3D structure homology analysis (Spalog). We found a panel of 25 and 26 upregulated proteins in the epimastigote and metacyclic trypomastigote stages, respectively; among these, 18 CHPs (8 in the epimastigote stage and 10 in the metacyclic stage) were characterized. The data generated corroborate the literature and complement the functional analyses of differentially regulated proteins at each stage, as they attribute potential functions to CHPs, which are frequently identified in Trypanosoma cruzi proteomics studies. However, it is important to point out that experimental validation is required to deepen our understanding of the CHPs.

Complement System Activation Is a Plasma Biomarker Signature during Malaria in Pregnancy.

Malaria in pregnancy (MiP) is a public health problem in malaria-endemic areas, contributing to detrimental outcomes for both mother and fetus. Primigravida and second-time mothers are most affected by severe anemia complications and babies with low birth weight compared to multigravida women. Infected erythrocytes (IE) reach the placenta, activating the immune response by placental monocyte infiltration and inflammation. However, specific markers of MiP result in poor outcomes, such as low birth weight, and intrauterine growth restriction for babies and maternal anemia in women infected with Plasmodium falciparum are limited. In this study, we identified the plasma proteome signature of a mouse model infected with Plasmodium berghei ANKA and pregnant women infected with Plasmodium falciparum infection using quantitative mass spectrometry-based proteomics. A total of 279 and 249 proteins were quantified in murine and human plasma samples, of which 28% and 30% were regulated proteins, respectively. Most of the regulated proteins in both organisms are involved in complement system activation during malaria in pregnancy. CBA anaphylatoxin assay confirmed the complement system activation by the increase in C3a and C4a anaphylatoxins in the infected plasma compared to non-infected plasma. Moreover, correlation analysis showed the association between complement system activation and reduced head circumference in newborns from Pf-infected mothers. The data obtained in this study highlight the correlation between the complement system and immune and newborn outcomes resulting from malaria in pregnancy.

Deciphering the Functional Status of Breast Cancers through the Analysis of Their Extracellular Vesicles.

Breast cancer (BC) accounts for the highest incidence of tumor-related mortality among women worldwide, justifying the growing search for molecular tools for the early diagnosis and follow-up of BC patients under treatment. Circulating extracellular vesicles (EVs) are membranous nanocompartments produced by all human cells, including tumor cells. Since minimally invasive methods collect EVs, which represent reservoirs of signals for cell communication, these particles have attracted the interest of many researchers aiming to improve BC screening and treatment. Here, we analyzed the cargoes of BC-derived EVs, both proteins and nucleic acids, which yielded a comprehensive list of potential markers divided into four distinct categories, namely, (i) modulation of aggressiveness and growth; (ii) preparation of the pre-metastatic niche; (iii) epithelial-to-mesenchymal transition; and (iv) drug resistance phenotype, further classified according to their specificity and sensitivity as vesicular BC biomarkers. We discuss the therapeutic potential of and barriers to the clinical implementation of EV-based tests, including the heterogeneity of EVs and the available technologies for analyzing their content, to present a consistent, reproducible, and affordable set of markers for further evaluation.

GBM Cells Exhibit Susceptibility to Metformin Treatment According to TLR4 Pathway Activation and Metabolic and Antioxidant Status.

Glioblastoma (GBM) is an aggressive brain cancer associated with poor overall survival. The metabolic status and tumor microenvironment of GBM cells have been targeted to improve therapeutic strategies. TLR4 is an important innate immune receptor capable of recognizing pathogens and danger-associated molecules. We have previously demonstrated the presence of TLR4 in GBM tumors and the decreased viability of the GBM tumor cell line after lipopolysaccharide (LPS) (TLR4 agonist) stimulation. In the present study, metformin (MET) treatment, used in combination with temozolomide (TMZ) in two GBM cell lines (U87MG and A172) and stimulated with LPS was analyzed. MET is a drug widely used for the treatment of diabetes and has been repurposed for cancer treatment owing to its anti-proliferative and anti-inflammatory actions. The aim of the study was to investigate MET and LPS treatment in two GBM cell lines with different metabolic statuses. MET treatment led to mitochondrial respiration blunting and oxidative stress with superoxide production in both cell lines, more markedly in U87MG cells. Decreased cell viability after MET + TMZ and MET + LPS + TMZ treatment was observed in both cell lines. U87MG cells exhibited apoptosis after MET + LPS + TMZ treatment, promoting increased ER stress, unfolded protein response, and BLC2 downregulation. LPS stimulation of U87MG cells led to upregulation of SOD2 and genes related to the TLR4 signaling pathway, including IL1B and CXCL8. A172 cells attained upregulated antioxidant gene expression, particularly SOD1, TXN and PRDX1-5, while MET treatment led to cell-cycle arrest. In silico analysis of the TCGA-GBM-RNASeq dataset indicated that the glycolytic plurimetabolic (GPM)-GBM subtype had a transcriptomic profile which overlapped with U87MG cells, suggesting GBM cases exhibiting this metabolic background with an activated inflammatory TLR4 pathway may respond to MET treatment. For cases with upregulated CXCL8, coding for IL8 (a pro-angiogenic factor), combination treatment with an IL8 inhibitor may improve tumor growth control. The A172 cell line corresponded to the mitochondrial (MTC)-GBM subtype, where MET plus an antioxidant inhibitor, such as anti-SOD1, may be indicated as a combinatory therapy.

Protein Arginylation Is Regulated during SARS-CoV-2 Infection.

BACKGROUND: In 2019, the world witnessed the onset of an unprecedented pandemic. By February 2022, the infection by SARS-CoV-2 has already been responsible for the death of more than 5 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER stress and activation of the unfolded protein response (UPR) pathway. Degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-degron pathway. RESULTS: The role of protein arginylation during SARS-CoV-2 infection was elucidated. Protein arginylation was studied in Vero CCL-81, macrophage-like THP1, and Calu-3 cells infected at different times. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure levels of arginyl-tRNA-protein transferase (ATE1) and its substrates. Dysregulation of the N-degron pathway was specifically identified during coronavirus infections compared to other respiratory viruses. We demonstrated that during SARS-CoV-2 infection, there is an increase in ATE1 expression in Calu-3 and Vero CCL-81 cells. On the other hand, infected macrophages showed no enzyme regulation. ATE1 and protein arginylation was variant-dependent, as shown using P1 and P2 viral variants and HEK 293T cells transfection with the spike protein and receptor-binding domains (RBD). In addition, we report that ATE1 inhibitors, tannic acid and merbromine (MER) reduce viral load. This finding was confirmed in ATE1-silenced cells. CONCLUSIONS: We demonstrate that ATE1 is increased during SARS-CoV-2 infection and its inhibition has potential therapeutic value.

Aberrant Protein Glycosylation in Brain Cancers, with Emphasis on Glioblastoma.

Aberrant glycosylation has been associated with several processes of tumorigenesis from cell signaling, migration and invasion, to immune regulation and metastasis formation. The biosynthesis of glycoconjugates is regulated through concerted and finely tuned enzymatic reactions. This includes the levels and activity of glycosyltransferases and glycosidases, nucleotide sugar metabolism, substrate availability, epigenetic condition, and cellular functional state. Glioblastoma (GBM) is the most aggressive brain tumor, frequently occurring in adults with overall survival not surpassing 17 months after diagnosis. GBM has been classified by the World Health Organization (WHO) as a grade 4 astrocytoma and stratified into G-CIMP, proneural, classical, and mesenchymal subtypes. Several biomolecular features associated with GBM aggressiveness have been elucidated; however, more studies are needed to elucidate the role of glycosylation in GBM pathology, looking at their potential as cancer targets. Here, we focus on the alteration of genes involved in protein N- and O-linked glycosylation in GBM. Specifically, the mRNA levels of glycogenes were analyzed using astrocytoma-TCGA-RNAseq datasets from public repositories. A total of 68 genes were differentially regulated in the most aggressive, mesenchymal subtype of GBM compared to the proneural and classical subtypes, and the expression of these genes was compared to normal brain tissues. Among them, we focused on 38 genes coding for proteins that belong to: 1) asparagine glycosylation (ALG); 2) glycosyltransferases (B3T, B4T); 3) fucosyltransferase (FUT); 4) acetylgalactosaminyltransferases (GALNT); 5) hexosaminidase (HEX); 6) mannosidase (MAN); 7) acetylglucosaminyltransferase (MGAT); 8) sialidase or neuraminidase (NEU); 9) solute carrier 35 family (SLC); and 10) sialyltransferase (ST). The differential expression of some genes was already reported in several solid tumors; however, several of them were found to be dysregulated in GBM for the first time. These data represent an important starting point to perform further orthogonal and functional validations to pinpoint the role of these glycogenes in GBM as diagnostic and therapeutic targets.

Glycoprotein molecular dynamics analysis: SARS-CoV-2 spike glycoprotein case study.

Molecular Dynamics (MD) is a method used to calculate the movement of atoms and molecules broadly applied to several aspects of science. It involves computational simulation, which makes it, at first glance, not easily accessible. The rise of several automated tools to perform molecular simulations has allowed researchers to navigate through the various steps of MD. This enables to elucidate structural properties of proteins that could not be analyzed otherwise, such as the impact of glycosylation. Glycosylation dictates the physicochemical and biological properties of a protein modulating its solubility, stability, resistance to proteolysis, interaction partners, enzymatic activity, binding and recognition. Given the high conformational and compositional diversity of the glycan chains, assessing their influence on the protein structure is challenging using conventional analytical techniques. In this manuscript, we present a step-by-step workflow to build and perform MD analysis of glycoproteins focusing on the SPIKE glycoprotein of SARS-CoV-2 to appraise the impact of glycans in structure stabilization and antibody occlusion.

Exploring COVID-19 pathogenesis on command-line: A bioinformatics pipeline for handling and integrating omics data.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in late 2019 in Wuhan, China, and has proven to be highly pathogenic, making it a global public health threat. The immediate need to understand the mechanisms and impact of the virus made omics techniques stand out, as they can offer a holistic and comprehensive view of thousands of molecules in a single experiment. Mastering bioinformatics tools to process, analyze, integrate, and interpret omics data is a powerful knowledge to enrich results. We present a robust and open access computational pipeline for extracting information from quantitative proteomics and transcriptomics public data. We present the entire pipeline from raw data to differentially expressed genes. We explore processes and pathways related to mapped transcripts and proteins. A pipeline is presented to integrate and compare proteomics and transcriptomics data using also packages available in the Bioconductor and providing the codes used. Cholesterol metabolism, immune system activity, ECM, and proteasomal degradation pathways increased in infected patients. Leukocyte activation profile was overrepresented in both proteomics and transcriptomics data. Finally, we found a panel of proteins and transcripts regulated in the same direction in the lung transcriptome and plasma proteome that distinguish healthy and infected individuals. This panel of markers was confirmed in another cohort of patients, thus validating the robustness and functionality of the tools presented.

Urine proteomics as a non-invasive approach to monitor exertional rhabdomyolysis during military training.

Exertional rhabdomyolysis (ERM), a condition often associated with strenuous exercise, a common practice in the military activities, can be defined as the process of injury and rupture of muscle cell membranes, with leakage of its components into the bloodstream. Creatine kinase (CK) has been used for ERM diagnosis, albeit several studies reported the discrepancy between CK levels and clinical signs or symptoms. In this study, we analyzed the biochemical profile of the blood, and the urinary proteome of ten marine soldiers in a special training course. The samples were collected in two periods, M1 and M2, which correspond to the lowest and highest CK levels during training, respectively. Quantitative urinary proteome profile of M1 and M2 showed changes in proteins involved in immune system and cell adhesion-related pathways after strenuous physical exercise. Changes in the abundance of several proteins was observed in individuals carrying genetic polymorphisms related to greater risk for muscle damage. A panel of proteins (CTSH, PIK3IP1, DEFB1, ITGB1, BCAN, and TNFRSF10C) presented high correlation with classical blood biochemical markers of ERM and AGT MET235Thr and ACE I/D polymorphisms. These proteins represent potential urine markers of muscle damage due to intense physical conditions such as military training activities. SIGNIFICANCE: This study analyzed the blood and urine of a cohort of marine soldiers enrolled in a special training program including missions with low and high exposure to strenuous exercise. The biochemical blood profile, polymorphisms mapping and mass spectrometry-based analyses of the urinary proteome was evaluated in such a controlled samples. A total of 226 urinary proteins associated to immune system, cell adhesion and redox homeostasis were significantly changes during ERM shedding lights on the disease pathogenesis. In particular, a panel of six proteins were associated to classical ERM markers and could be used as early non invasive biomarkers.

Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay.

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. SIGNIFICANCE: We describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes.

Risk factors and future directions for preventing and diagnosing exertional rhabdomyolysis.

Exertional rhabdomyolysis may occur when an individual is subjected to strenuous physical exercise. It is occasionally associated with myoglobinuria (i.e. "cola-colored" urine) alongside muscle pain and weakness. The pathophysiology of exertional rhabdomyolysis involves striated muscle damage and the release of cellular components into extracellular fluid and bloodstream. This can cause acute renal failure, electrolyte abnormalities, arrhythmias and potentially death. Exertional rhabdomyolysis is observed in high-performance athletes who are subjected to intense, repetitive and/or prolonged exercise but is also observed in untrained individuals and highly trained or elite groups of military personnel. Several risk factors have been reported to increase the likelihood of the condition in athletes, including: viral infection, drug and alcohol abuse, exercise in intensely hot and humid environments, genetic polymorphisms (e.g. sickle cell trait and McArdle disease) and epigenetic modifications. This article reviews several of these risk factors and proposes screening protocols to identify individual susceptibility to exertional rhabdomyolysis as well as the relevance of proteomics for the evaluation of potential biomarkers of muscle damage.

HSPB1 influences mitochondrial respiration in ER-stressed beta cells.

Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.

Protein glycosylation in extracellular vesicles: Structural characterization and biological functions.

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles involved in intercellular communication, delivery of biomolecules from donor to recipient cells, cellular disposal and homeostasis, potential biomarkers and drug carriers. The content of EVs includes DNA, lipids, metabolites, proteins, and microRNA, which have been studied in various diseases, such as cancer, diabetes, pregnancy, neurodegenerative, and cardiovascular disorders. EVs are enriched in glycoconjugates and exhibit specific glycosignatures. Protein glycosylation is a co- and post-translational modification (PTM) that plays an important role in the expression and function of exosomal proteins. N- and O-linked protein glycosylation has been mapped in exosomal proteins. The purpose of this review is to highlight the importance of glycosylation in EVs proteins. Initially, we describe the main PTMs in EVs with a focus on glycosylation. Then, we explore glycan-binding proteins describing the main findings of studies that investigated the glycosylation of EVs in cancer, pregnancy, infectious diseases, diabetes, mental disorders, and animal fluids. We have highlighted studies that have developed innovative methods for studying the content of EVs. In addition, we present works related to lipid glycosylation. We explored the content of studies deposited in public databases, such as Exocarta and Vesiclepedia. Finally, we discuss analytical methods for structural characterization of glycoconjugates and present an overview of the critical points of the study of glycosylation EVs, as well as perspectives in this field.

Serum Proteomics Reveals Alterations in Protease Activity, Axon Guidance, and Visual Phototransduction Pathways in Infants With In Utero Exposure to Zika Virus Without Congenital Zika Syndrome.

In 2015, ZIKV infection attracted international attention during an epidemic in the Americas, when neurological disorders were reported in infants who had their mothers exposed to ZIKV during pregnancy. World Health Organization (WHO) epidemiological data show that 5 to 15% of neonates exposed to ZIKV in the uterus have complications included in abnormalities related to Congenital Zika Syndrome (CZS). The risk of complications after birth is not well documented, however, clinical evidence shows that 6% of infants exposed to ZIKV during pregnancy have complications present at birth, and this rate rises to 14% when medical monitoring is performed in all exposed infants, regardless of birth condition. Thus, the evaluation and monitoring of all exposed infants are of foremost importance as the development of late complications has been increasingly supported by clinical evidence. The identification of changes in protein profile of infants exposed to ZIKV without CZS could provide valuable findings to better understand molecular changes in this cohort. Here, we use a shotgun-proteomics approach to investigate alterations in the serum of infants without CZS symptoms but exposed to intrauterine ZIKV (ZIKV) compared to unexposed controls (CTRL). A complex pattern of differentially expressed proteins was identified, highlighting the dysregulation of proteins involved in axon orientation, visual phototransduction, and global protease activity in children exposed to ZIKV without CZS. These data support the importance of monitoring children exposed to ZIKV during gestation and without early CZS symptoms. Our study is the first to assess molecular evidence of possible late disorders in children victims of the ZIKV outbreak in the Americas. We emphasize the importance of medical monitoring of symptomatic and asymptomatic children, as apparently unexplained late neurological and eye disorders may be due to intrauterine ZIKV exposure.

Lights and Shadows of TORCH Infection Proteomics.

Congenital abnormalities cause serious fetal consequences. The term TORCH is used to designate the most common perinatal infections, where: (T) refers to toxoplasmosis, (O) means "others" and includes syphilis, varicella-zoster, parvovirus B19, zika virus (ZIKV), and malaria among others, (R) refers to rubella, (C) relates to cytomegalovirus infection, and (H) to herpes simplex virus infections. Among the main abnormalities identified in neonates exposed to congenital infections are central nervous system (CNS) damage, microcephaly, hearing loss, and ophthalmological impairment, all requiring regular follow-up to monitor its progression. Protein changes such as mutations, post-translational modifications, abundance, structure, and function may indicate a pathological condition before the onset of the first symptoms, allowing early diagnosis and understanding of a particular disease or infection. The term "proteomics" is defined as the science that studies the proteome, which consists of the total protein content of a cell, tissue or organism in a given space and time, including post-translational modifications (PTMs) and interactions between proteins. Currently, quantitative bottom-up proteomic strategies allow rapid and high throughput characterization of complex biological mixtures. Investigating proteome modulation during host-pathogen interaction helps in elucidating the mechanisms of infection and in predicting disease progression. This "molecular battle" between host and pathogen is a key to identify drug targets and diagnostic markers. Here, we conducted a survey on proteomic techniques applied to congenital diseases classified in the terminology "TORCH", including toxoplasmosis, ZIKV, malaria, syphilis, human immunodeficiency virus (HIV), herpes simplex virus (HSV) and human cytomegalovirus (HCVM). We have highlighted proteins and/or protein complexes actively involved in the infection. Most of the proteomic studies reported have been performed in cell line models, and the evaluation of tissues (brain, muscle, and placenta) and biofluids (plasma, serum and urine) in animal models is still underexplored. Moreover, there are a plethora of studies focusing on the pathogen or the host without considering the triad mother-fetus-pathogen as a dynamic and interconnected system.