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Current clinical tools for breast cancer (BC) diagnosis are insufficient but liquid biopsy of different bodily fluids has recently emerged as a minimally invasive strategy that provides a real-time snapshot of tumour biomarkers for early diagnosis, active surveillance of progression, and post-treatment recurrence. Extracellular vesicles (EVs) are nano-sized membranous structures 50–1000 nm in diameter that are released by cells into biological fluids. EVs contain proteins, nucleic acids, and lipids which play pivotal roles in tumourigenesis and metastasis through cell-to-cell communication. Proteins and miRNAs from small EVs (sEV), which range in size from 50–150 nm, are being investigated as a potential source for novel BC biomarkers using mass spectrometry-based proteomics and next-generation sequencing. This review covers recent developments in sEV isolation and single sEV analysis technologies and summarises the sEV protein and miRNA biomarkers identified for BC diagnosis, prognosis, and chemoresistance. The limitations of current sEV biomarker research are discussed along with future perspective applications.

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression. We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20). Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively. Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.

Background Serum protein biomarkers are used to diagnose, monitor treatment response, and to differentiate various forms of chronic enteropathies (CE) in humans. The utility of liquid biopsy proteomic approaches has not been examined in cats. Hypothesis/Objectives To explore the serum proteome in cats to identify markers differentiating healthy cats from cats with CE. Animals Ten cats with CE with signs of gastrointestinal disease of at least 3 weeks duration, and biopsy‐confirmed diagnoses, with or without treatment and 19 healthy cats were included. Methods Cross‐sectional, multicenter, exploratory study with cases recruited from 3 veterinary hospitals between May 2019 and November 2020. Serum samples were analyzed and evaluated using mass spectrometry‐based proteomic techniques. Results Twenty‐six proteins were significantly (P < .02, ≥5‐fold change in abundance) differentially expressed between cats with CE and controls. Thrombospondin‐1 (THBS1) was identified with >50‐fold increase in abundance in cats with CE (P < 0.001) compared to healthy cats. Conclusions and Clinical Importance Damage to the gut lining released marker proteins of chronic inflammation that were detectable in serum samples of cats. This early‐stage exploratory study strongly supports THBS1 as a candidate biomarker for chronic inflammatory enteropathy in cats.

Background Chronic enteropathy (CE) is common in dogs and can occur with multiple etiologies including food‐responsive enteropathy (FRE) and idiopathic inflammatory bowel disease (IBD). Hypothesis/Objective To study the protein profile and pathway differences among dogs with FRE, IBD, and healthy controls using serum proteome analysis. Animals Nine CE dogs with signs of gastrointestinal disease and histologically confirmed chronic inflammatory enteropathy and 16 healthy controls. Methods A cross‐sectional study with cases recruited from 2 veterinary hospitals between May 2019 and November 2020 was performed. Serum samples were analyzed using mass spectrometry‐based proteomic techniques. Results Proteomic profiles showed marked variation in relative protein abundances. Forty‐five proteins were significantly (P ≤ .01) differentially expressed among the dogs with CE and controls with ≥2‐fold change in abundance. The fold change of dogs with IBD normalized to controls was more pronounced for the majority of proteins than that seen in the dogs with FRE normalized to control dogs. Proteins involving reactive oxygen species, cytokine activation, acute phase response signaling, and lipid metabolism were altered in dogs with CE. Conclusions and Clinical Importance Cytokine alterations, acute phase response signaling, and lipid metabolism are likely involved in pathogenesis of CE. Although there are insufficient current data to justify the use of proteomic biomarkers for assessment of CE in dogs, our study identifies potential candidates.

Over the years, the scientific community has explored myriads of theories in search of the etiology and a cure for inflammatory bowel disease (IBD). The cumulative evidence has pointed to the key role of the intestinal barrier and the breakdown of these mechanisms in IBD. More and more scientists and clinicians are embracing the concept of the impaired intestinal epithelial barrier and its role in the pathogenesis and natural history of IBD. However, we are missing a key tool that bridges these scientific insights to clinical practice. Our goal is to overcome the limitations in understanding the molecular physiology of intestinal barrier function and develop a clinical tool to assess and quantify it. This review article explores the proteins in the intestinal tissue that are pivotal in regulating intestinal permeability. Understanding the molecular pathophysiology of impaired intestinal barrier function in IBD may lead to the development of a biochemical method of assessing intestinal tissue integrity which will have a significant impact on the development of novel therapies targeting the intestinal mucosa.

Small extracellular vesicles (sEVs) are an important intercellular communicator, participating in all stages of cancer metastasis, immunity, and therapeutic resistance. Therefore, protein cargoes within sEVs are considered as a superior source for breast cancer (BC) biomarker discovery. Our study aimed to optimise the approach for sEV isolation and sEV proteomic analysis to identify potential sEV protein biomarkers for BC diagnosis. sEVs derived from BC cell lines, BC patients’ plasma, and non-cancer controls were isolated using ultracentrifugation (UC), a Total Exosome Isolation kit (TEI), and a combined approach named UCT. In BC cell lines, the UC isolates showed a higher sEV purity and marker expression, as well as a higher number of sEV proteins. In BC plasma samples, the UCT isolates showed the highest proportion of sEV-related proteins and the lowest percentage of lipoprotein-related proteins. Our data suggest that the assessment of both the quantity and quality of sEV isolation methods is important in selecting the optimal approach for the specific sEV research purpose, depending on the sample types and downstream analysis.

The epithelial barrier’s primary role is to protect against entry of foreign and pathogenic elements. Both COVID-19 and Inflammatory Bowel Disease (IBD) show commonalities in symptoms and treatment with sensitization of the epithelial barrier inviting an immune response. In this study we use a multi-omics strategy to identify a common signature of immune disease that may be able to predict for more severe patient outcomes. Global proteomic approaches were applied to transcriptome and proteome. Further semi- and relative- quantitative targeted mass spectrometry methods were developed to substantiate the proteomic and metabolomics changes in nasal swabs from healthy, COVID-19 (24 h and 3 weeks post infection); serums from Crohn’s disease patients (scored for epithelial leak), terminal ileum tissue biopsies (patient matched inflamed and non-inflamed regions, and controls). We found that the tryptophan/kynurenine metabolism pathway is a ‘hub’ regulator of canonical and non-canonical transcription, macrophage release of cytokines and significant changes in the immune and metabolic status with increasing severity and disease course. Significantly modified pathways include stress response regulator EIF2 signaling (p = 1 × 10−3); energy metabolism, KYNU (p = 4 × 10−4), WARS (p = 1 × 10−7); inflammation, and IDO activity (p = 1 × 10−6). Heightened levels of PARP1, WARS and KYNU are predictive at the acute stage of infection for resilience, while in contrast, levels remained high and are predictive of persistent and more severe outcomes in COVID disease. Generation of a targeted marker profile showed these changes in immune disease underlay resolution of epithelial barrier function and have the potential to define disease trajectory and more severe patient outcomes.

Identifying biomarkers and signaling pathways are important for the management of prostate cancer (CaP) radioresistance. In this study, we identified differential proteins and signaling pathways from parental CaP cell lines and CaP radioresistant (RR) sublines using a label-free LC-MS/MS proteomics approach. A total of 309 signaling pathway proteins were identified to be significantly altered between CaP and CaP-RR cells ( p  ≤ 0.05, fold differences >1.5, ≥80% power). Among these proteins, nineteen are common among three paired CaP cell lines and associated with metastasis, progression and radioresistance. The PI3K/Akt, VEGF and glucose metabolism pathways were identified as the main pathways associated with CaP radioresistance. In addition, the identified potential protein markers were further validated in CaP-RR cell lines and subcutaneous (s.c) animal xenografts by western blotting and immunohistochemistry, respectively and protein aldolase A (ALDOA) was selected for a radiosensitivity study. We found the depletion of ALDOA combined with radiotherapy effectively reduced colony formation, induced more apoptosis and increased radiosensitivity in CaP-RR cells. Our findings indicate that CaP radioresistance is caused by multifactorial traits and downregulation of ALDOA increases radiosensitivity in CaP-RR cells, suggesting that controlling these identified proteins or signaling pathways in combination with radiotherapy may hold promise to overcome CaP radioresistance.

The transitioning of cells during the systemic demise of an organism is poorly understood. Here, we present evidence that organismal death is accompanied by a common and sequential molecular flood of stress-induced events that propagate the senescence phenotype, and this phenotype is preserved in the proteome after death. We demonstrate activation of “death” pathways involvement in diseases of ageing, with biochemical mechanisms mapping onto neurological damage, embryonic development, the inflammatory response, cardiac disease and ultimately cancer with increased significance. There is sufficient bioavailability of the building blocks required to support the continued translation, energy, and functional catalytic activity of proteins. Significant abundance changes occur in 1258 proteins across 1 to 720 h post-mortem of the 12-week-old mouse mandible. Protein abundance increases concord with enzyme activity, while mitochondrial dysfunction is evident with metabolic reprogramming. This study reveals differences in protein abundances which are akin to states of stress-induced premature senescence (SIPS). The control of these pathways is significant for a large number of biological scenarios. Understanding how these pathways function during the process of cellular death holds promise in generating novel solutions capable of overcoming disease complications, maintaining organ transplant viability and could influence the findings of proteomics through “deep-time” of individuals with no historically recorded cause of death.

The glutathione shunt is one of the most important contributors to the cellular redox state, with implications across cancer, chronic diseases, diseases of ageing, and autoimmune diseases, including inflammatory bowel disease (IBD). Traditionally, the redox state is gauged by the ratio of the surrogate metabolites GSH and GSSG. However, this presents methodological challenges and offers a constrained illustration of metabolites without a systems-level understanding of redox dynamics, failing to elucidate variations across an entire biochemical network. Targeted proteomics can fill this void. Here, we describe an in-parallel metabolomic and proteomic targeted method to encompass measurements directly related to the shunt. Samples are simultaneously prepared to extract the substrate building blocks, cysteine, cystine, methionine, glutamic acid, and kynurenine; and the proteins, SLC7A11 (xCT), Glutamate Cysteine Ligase (GSH1), Glutathione Synthetase (GSH2), Glutathione Peroxidase (GPx), and Glutathione Reductase (GSHR) for targeted mass spectrometry. We demonstrate the method by targeted analysis of proteins in plasma, serum, nasal swab, and saliva and apply the multi-omic method to assess changes in the glutathione shunt in the serum of patients diagnosed with IBD. This allows for a broader narrative to establish context at which the glutathione shunt is operating.