Peptidomics and machine-learning-based evaluation of ncRNA-derived micropeptides in breast cancer: Expression patterns and functional/therapeutic insights.

Breast cancer is a highly heterogeneous disease characterized by different subtypes arising from molecular alterations that give the disease different phenotypes, clinical behaviors, and prognostic. The ncRNA-derived micropeptides (MPs) represent a novel layer of complexity in cancer study once they can be biologically active and can present potential as biomarkers and also in therapeutics. However, few large-scale studies address the expression of these peptides at the peptidomics level or evaluate their functions and potential in peptide-based therapeutics for breast cancer. In this study, we propose deepening the landscape of ncRNA-derived MPs in breast cancer subtypes and advance the comprehension of the relevance of these molecules to the disease. Firstly, we constructed a 16,349 unique putative MP sequence dataset by integrating two previously published lists of predicted ncRNA-derived MPs. We evaluated its expression on high-throughput mass spectrometry data of breast tumor samples from different subtypes. Next, we applied several machine and deep learning tools, such as AntiCP 2.0, MULocDeep, PEPstrMOD, Peptipedia, and PreAIP, to predict its functions, cellular localization, tertiary structure, physicochemical features, and other properties related to therapeutics. We identified 58 peptides expressed on breast tissue, including 27 differentially expressed MPs in tumor compared to non-tumor samples and MPs exhibiting tumor or subtype specificity. These peptides presented physicochemical features compatible with the canonical proteome and were predicted to influence the tumor immune environment and participate in cell communication, metabolism, and signaling processes. Also, some MPs presented potential as anti-cancer, anti-inflammatory, and anti-angiogenic molecules. Our data demonstrate that MPs derived from ncRNAs have expression patterns associated with specific breast cancer subtypes and tumor specificity, thus highlighting their potential as biomarkers for molecular classification. We also reinforce the relevance of MPs as biologically active molecules that play a role in breast tumorigenesis, besides their potential in peptide-based therapeutics.

A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines.

RATIONALE: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. METHODS: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. RESULTS: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. CONCLUSIONS: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.

Non-histone protein methylation in Trypanosoma cruzi epimastigotes.

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.

Screening of Exosome-Derived Proteins and Their Potential as Biomarkers in Diagnostic and Prognostic for Pancreatic Cancer.

In the oncological area, pancreatic cancer is one of the most lethal diseases, with 5-year survival rising just 10% in high-development countries. This disease is genetically characterized by KRAS as a driven mutation followed by SMAD4, CDKN2, and TP53-associated mutations. In clinical aspects, pancreatic cancer presents unspecific clinical symptoms with the absence of screening and early plasmatic biomarker, being that CA19-9 is the unique plasmatic biomarker having specificity and sensitivity limitations. We analyzed the plasmatic exosome proteomic profile of 23 patients with pancreatic cancer and 10 healthy controls by using Nanoscale liquid chromatography coupled to tandem mass spectrometry (NanoLC-MS/MS). The pancreatic cancer patients were subdivided into IPMN and PDAC. Our findings show 33, 34, and 7 differentially expressed proteins when comparing the IPMN vs. control, PDAC-No treatment vs. control, and PDAC-No treatment vs. IPMN groups, highlighting proteins of the complement system and coagulation, such as C3, APOB, and SERPINA. Additionally, PDAC with no treatment showed 11 differentially expressed proteins when compared to Folfirinox neoadjuvant therapy or Gemcitabine adjuvant therapy. So here, we found plasmatic exosome-derived differentially expressed proteins among cancer patients (IPMN, PDAC) when comparing with healthy controls, which could represent alternative biomarkers for diagnostic and prognostic evaluation, supporting further scientific and clinical studies on pancreatic cancer.

Functional EGF domain of the human neuregulin 1α produced in Escherichia coli with accurate disulfide bonds.

BACKGROUND: Neuregulins comprise a large family of growth factors containing an epidermal growth factor (EGF) domain. NRG1 acts in signaling pathways involved in proliferation, apoptosis, migration, differentiation, and adhesion of many normal cell types and in human diseases. The EGF domain of NRG1 mediates signaling by interaction with members of the ErbB family of receptors. Easy access to correctly folded hNRG1α EGF domain can be a valuable tool to investigate its function in different cell types. MATERIALS AND METHODS: The EGF domain of hNRG1α was produced in Escherichia coli in fusion with TrxA and purified after cleavage of TrxA. Conformation and stability analyses were performed by using biophysical methods and the disulfide bonds were mapped by mass spectrometry. The activity of the hNRG1α EGF domain was demonstrated in cell proliferation and migration assays. RESULTS: Approximately 3.3 mg of hNRG1α EGF domain were obtained starting from a 0.5 L of E. coli culture. Correct formation of the three disulfide bonds was demonstrated by mass spectrometry with high accuracy. Heat denaturation assays monitored by circular dichroism and dynamic light scattering revealed that it is a highly stable protein. The recombinant EGF domain of hNRG1α purified in this work is highly active, inducing cell proliferation at concentration as low as 0.05 ng/mL. It induces also cell migration as demonstrated by a gap closure assay. CONCLUSION: The EGF domain of hNRG1α was produced in E. coli with the correct disulfide bonds and presented high stimulation of HeLa cell proliferation and NDFH cell migration.

A proteomics evaluation of the primary and secondary immune response of Biomphalaria straminea challenged by Schistosoma mansoni.

Biomphalaria spp. snails are intermediary hosts of Schistosoma mansoni, etiologic agent of intestinal schistosomiasis, one of the most important neglected tropical diseases. Biomphalaria straminea is an important intermediary host that possess a different phenotype to parasite infection but shows a large geographic distribution and high capacity of new ecologic niche invasion. Our purpose was to characterize for the first time the differentially expressed proteome in B. straminea during two times intervals after primary and secondary exposure to S. mansoni. The hemolymph was collected at 1 and 15 days after primary and secondary exposure of snails to the parasite. Total proteins were extracted and digested with trypsin. LC-MS/MS label-free quantification was performed and analyzed using Maxquant and Perseus software. Proteins were identified and annotated using Blast2GO tools. After 1 day of exposure, most of upregulated proteins are hemoglobin type 2, C and H type lectins, molecules related to cell adhesion, and response to oxidative stress. After 15 days, we found a similar pattern of upregulated proteins but some fibrinogen-related proteins (FREPs) and TEPs homologs were downregulated. Regarding the differentially expressed proteins during secondary response, the principal immune-related proteins upregulated were C and H type lectins, cellular adhesion molecules, biomphalysin, and FREP3. We noted a several upregulated biological processes during both responses that could be the one of the key points of efficacy in the immune response to parasite. Our data suggests different immune mechanisms used by B. straminea snails challenged with S. mansoni.

Integrated analysis of label-free quantitative proteomics and bioinformatics reveal insights into signaling pathways in male breast cancer.

Male breast cancer (MBC) is a rare malignancy that accounts for about 1.8% of all breast cancer cases. In contrast to the high number of the "omics" studies in breast cancer in women, only recently molecular approaches have been performed in MBC research. High-throughput proteomics based methodologies are promisor strategies to characterize the MBC proteomic signatures and their association with clinico-pathological parameters. In this study, the label-free quantification-mass spectrometry and bioinformatics approaches were applied to analyze the proteomic profiling of a MBC case using the primary breast tumor and the corresponding axillary metastatic lymph nodes and adjacent non-tumor breast tissues. The differentially expressed proteins were identified in the signaling pathways of granzyme B, sirtuins, eIF2, actin cytoskeleton, eNOS, acute phase response and calcium and were connected to the upstream regulators MYC, PI3K SMARCA4 and cancer-related chemical drugs. An additional proteomic comparative analysis was performed with a primary breast tumor of a female patient and revealed an interesting set of proteins, which were mainly involved in cancer biology. Together, our data provide a relevant data source for the MBC research that can help the therapeutic strategies for its management.

Characterising ISWI chromatin remodeler in Trypanosoma cruzi.

BACKGROUND Imitation SWItch (ISWI) ATPase is the catalytic subunit in diverse chromatin remodeling complexes. These complexes modify histone-DNA interactions and therefore play a pivotal role in different DNA-dependent processes. In Trypanosoma cruzi, a protozoan that controls gene expression principally post-transcriptionally, the transcriptional regulation mechanisms mediated by chromatin remodeling are poorly understood. OBJECTIVE To characterise the ISWI remodeler in T. cruzi (TcISWI). METHODS A new version of pTcGW vectors was constructed to express green fluorescent protein (GFP)-tagged TcISWI. CRISPR-Cas9 system was used to obtain parasites with inactivated TcISWI gene and we determined TcISWI partners by cryomilling-affinity purification-mass spectrometry (MS) assay as an approximation to start to unravel the function of this protein. FINDINGS Our approach identified known ISWI partners [nucleoplasmin-like protein (NLP), regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP)], previously characterised in T. brucei, and new components in TcISWI complex [DRBD2, DHH1 and proteins containing a domain characteristic of structural maintenance of chromosomes (SMC) proteins]. Data are available via ProteomeXchange with identifier PXD017869. MAIN CONCLUSIONS In addition to its participation in transcriptional silencing, as it was reported in T. brucei, the data generated here provide a framework that suggests a role for TcISWI chromatin remodeler in different nuclear processes in T. cruzi, including mRNA nuclear export control and chromatin compaction. Further work is necessary to clarify the TcISWI functional diversity that arises from this protein interaction study.

Mitogen-Activated Protein Kinase Kinase 5 Regulates Proliferation and Biosynthetic Processes in Procyclic Forms of Trypanosoma brucei.

The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated protein kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light on the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acids biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acids biosynthesis in procyclic T. brucei.

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host.

Tissue-Derived Signals for Mesenchymal Stem Cell Stimulation: Role of Cardiac and Umbilical Cord Microenvironments.

The tissue microenvironment regulates such stem cell behaviors as self-renewal and differentiation. Attempts to mimic components of these microenvironments could provide new strategies for culturing and directing the behavior of stem cells. The aim of the present study was to mimic cardiac and umbilical cord tissue microenvironments in vitro and compare the resulting bone marrow-derived mesenchymal stem cell (BM-MSC) behaviors. We generated tissue microenvironments using conditioned medium (CM) and extracellular matrix (ECM) samples obtained from human heart and umbilical cord tissue explant cultures and by tissue decellularization. Mass spectrometry and immunostaining were used to characterize and determine the specific protein profiles of the ECMs and CMs. We demonstrated that the ECMs and CMs were not cytotoxic to BM-MSCs and could thus be tested via cell culture. The BM-MSCs showed a higher proliferation rate when cultured with umbilical cord-derived CM compared with the other analyzed conditions. Furthermore, the ECMs increased cell adhesion and migration. However, although the conditions tested in this work were able to maintain the viability and affect the proliferation, adhesion and migration of BM-MSCs in vitro, mimicking tissue microenvironments using ECM and CM was not sufficient to induce the cardiomyogenic differentiation of BM-MSCs. The present study provides a thorough characterization of the biological activity of these ECMs and CMs in human BM-MSC cultures.

Towards the phosphoproteome of trypanosomatids.

The identification and localization of protein phosphorylation sites provide clues to what proteins or pathways might be activated in a given condition, helping to improve our understanding about signaling networks. Advances in strategies for enrichment of phosphorylated peptides/proteins, mass spectrometry (MS) instrumentation, and specific MS techniques for identification and quantification of post-translational modifications have allowed for large-scale mapping of phosphorylation sites, promoting the field of phosphoproteomics. The great promise of phosphoproteomics is to unravel the dynamics of signaling networks, a layer of the emerging field of systems biology. Until a few years ago only a small number of phosphorylation sites had been described. Following large-scale trends, recent phosphoproteomic studies have reported the mapping of thousands of phosphorylation sites in trypanosomatids. However, quantitative information about the regulation of such sites in different conditions is still lacking. In this chapter, we provide a historical overview of phosphoproteomic studies for trypanosomatids and discuss some challenges and perspectives in the field.

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation.

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.

LM14 defined medium enables continuous growth of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between distinct morphological and functional forms during its life cycle. Axenic multiplication and differentiation processes of this protozoan parasite can be reproduced in vitro, enabling the isolation and study of the different evolutionary forms. Although there are several publications attempting the cultivation of T. cruzi under chemically defined conditions, in our experience none of the published media are capable of maintaining T. cruzi in continuous growth. RESULTS: In this work we modified a known chemically defined medium for Trypanosoma brucei growth. The resulting LM14 and LM14B defined media enabled cultivation of five different strains of T. cruzi for more than forty passages until now. The parasite's biological characteristics such as morphology and differentiation to metacyclic trypomastigotes were maintained when defined media is used. CONCLUSIONS: The establishment of a defined medium for T. cruzi cultivation is an important tool for basic biological research allowing several different approaches, providing new perspectives for further studies related to cell biology of this parasite.

A peptide dataset for target analysis of human complement system proteins.

The targeted LC-MS/MS method has been widely applied for peptide quantification, offering sensibility, specificity, and reproducibility to the analysis. However, it requires the prior selection of targets, including the construction of a spectral library. Here, we present a dataset comprising peptide mass spectra for targeted LC-MS/MS method setup, applied to a set of human complement system proteins. Additionally, we selected a group of peptides and demonstrated their stability and reproducibility in quantification. This dataset is invaluable for studies aiming at the quantification of the complement system proteins by targeted LC-MS/MS, as it provides data for spectral library construction and a list of selected peptides.

Hollow-fiber liquid phase microextraction for determination of fluoxetine in human serum by nano-liquid chromatography coupled to high resolution mass spectrometry.

Therapeutic drug monitoring (TDM) is a personalized care tool based on the determination of a target drug concentration in human serum. An antidepressant drug of interest for such investigations is fluoxetine (FXT), due to a severe impact of genetic polymorphisms on its metabolism. A bioanalytical method employed for TDM purposes must exhibit satisfactory selectivity and detectability, which becomes more difficult due to highly complex biological matrices. In this study, a highly selective bioanalytical method for the determination of FXT in human serum is proposed, which provides excellent clean-up efficiency based on a low cost hollow fiber liquid-phase microextraction (HF-LPME) sample preparation step and nano-liquid chromatography coupled to high-resolution mass spectrometry (nano-LC-HRMS). HF-LPME was performed using a two-phase "U" configuration, with 6 cm fiber, 20 µL of 1-octanol acting as supported liquid membrane, and ammonium hydroxide (pH 10) as the donor phase with NaCl (10 % m/v) and methanol (5 % v/v) as additives, requiring only 250 µL of the sample. The procedure was conducted for 30 min under a 750 rpm stirring rate. Gradient elution was carried out employing an acetonitrile-water as mobile phase, the composition of 30:70 to 100:00 (v/v) for 15 min, using formic acid 0.1 % (v/v) as an additive. MS1 was acquired in an Orbitrap mass analyzer, while MS2 was acquired in a linear trap quadrupole. Satisfactory linearity (Pearson's r = 0.99709) was obtained for a concentration range of 0.02 to 2.5 µg mL-1, which is compatible with the therapeutic and toxic range for FXT. The developed method presents adequate precision (1.61 to 7.45 %) and accuracy (95 to 114 %) and allows the dilution of high concentration samples in a 1:4 ratio (v/v), enabling its application for forensic serum samples. To our knowledge, this is the first study reporting a method based on HF-LPME and nano-LC-HRMS with any analytical purpose, especially with a TDM focus.

Unveiling novel targets in melanoma under melanogenesis stimulation and photodynamic therapy by redox proteomics.

Melanogenesis-stimulated B16-F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy (RB-PDT). A redox proteomics label-free approach based on the biotin switch assay technique with biotin-HPDP and N-ethylmaleimide was used to assess the thiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451, citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2) was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimulation. After RB-PDT, only guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation followed by RB-PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153 and Cys-249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) was oxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation followed by RB-PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms.

A dataset for developing proteomic tools for pathogen detection via differential cell lysis of whole blood samples.

This data descriptor presents a curated dataset for pathogen detection and identification (Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans) directly from whole-blood samples. The dataset was created using differential cell lysis combined with rapid extraction, digestion, and mass spectrometry-based proteomics. Our method offers a rapid diagnostic alternative to traditional culture, enabling timely disease management, such as sepsis. Highlighting our dataset's uniqueness, it features a three-tier structure: Spectral Libraries of Pathogens for identifying peptide peaks for putative biomarkers; Spiked pathogen in blood MS data for biomarker panel optimization through varied concentration samples; and Parallel Reaction Monitoring (PRM) data from sepsis patients for validating our biomarker panel, achieving 83.3% sensitivity within seven hours without microbial enrichment culture. This dataset serves as a comprehensive reference for bioinformatic tool development and biomarker panel proposals, advancing microbial detection, antimicrobial resistance, and epidemiological studies.

High-throughput proteomics of breast cancer subtypes: Biological characterization and multiple candidate biomarker panels to patients' stratification.

BACKGROUND AND AIMS: The actual classification of breast tumors in subtypes represents an attempt to stratify patients into clinically cohesive groups, nevertheless, clinicians still lack reproducible and reliable protein biomarkers for breast cancer subtype discrimination. In this study, we aimed to access the differentially expressed proteins between these tumors and its biological implications, contributing to the subtype's biological and clinical characterization, and with protein panels for subtype discrimination. METHODS: In our study, we applied high-throughput mass spectrometry, bioinformatic, and machine learning approaches to investigate the proteome of different breast cancer subtypes. RESULTS: We identified that each subtype depends on different protein expression patterns to sustain its malignancy, and also alterations in pathways and processes that can be associated with each subtype and its biological and clinical behaviors. Regarding subtype biomarkers, our panels achieved performances with at least 75% of sensibility and 92% of specificity. In the validation cohort, the panels obtained acceptable to outstanding performances (AUC = 0.740 to 1.00). CONCLUSIONS: In general, our results expand the accuracy of breast cancer subtypes' proteomic landscape and improve the understanding of its biological heterogeneity. In addition, we identified potential protein biomarkers for the stratification of breast cancer patients, improving the repertoire of reliable protein biomarkers. SIGNIFICANCE: Breast cancer is the most diagnosed cancer type worldwide and the most lethal cancer in women. As a heterogeneous disease, breast cancer tumors can be classified into four major subtypes, each presenting particular molecular alterations, clinical behaviors, and treatment responses. Thus, a pivotal step in patient management and clinical decisions is accurately classifying breast tumor subtypes. Currently, this classification is made by the immunohistochemical detection of four classical markers (estrogen receptor, progesterone receptor, HER2 receptor, and the Ki-67 index); however, it is known that these markers alone do not fully discriminate the breast tumor subtypes. Also, the poor understanding of the molecular alterations of each subtype leads to a challenging decision-making process regarding treatment choice and prognostic determination. This study, through high-throughput label-free mass-spectrometry data acquisition and downstream bioinformatic analysis, advances in the proteomic discrimination of breast tumors and achieves an in-depth characterization of the subtype's proteomes. Here, we indicate how the variations in the subtype's proteome can influence the tumor's biological and clinical differences, highlighting the variation in the expression pattern of oncoproteins and tumor suppressor proteins between subtypes. Also, through our machine-learning approach, we propose multi-protein panels with the potential to discriminate the breast cancer subtypes. Our panels achieved high classification performance in our cohort and in the independent validation cohort, demonstrating their potential to improve the current tumor discrimination system as complements to the classical immunohistochemical classification.

Proteomics and image screening data of cellular secretomes and their biological effects: Comparing the signals sent by cardiac stromal cells and dermal fibroblasts in culture.

The study of the secretome of different cell types has gained prominence over the years due to its role in understanding the cell microenvironment and possible uses in acellular therapies. Approaches in this field include proteomic characterizations of the secretomes as well as evaluating their potential to induce cell and tissue responses. Here, we present the mass spectrometry proteomics data from a characterization of the secretome of cardiac resident stromal cells (CRSCs) and dermal fibroblasts in order to compare their compositions. To evaluate the potential for cell proliferation, differentiation, migration, and adhesion, in vitro assays were performed and analyzed using a high-content imaging system. For each assay, specific analysis strategies were developed to quantify the generated data. These datasets provide insights into the differences and similarities between secretomes from different cell sources. It also describes methodologies for analyzing images from different in vitro assays using high-throughput automated imaging.