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This study evaluated the suitability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess enamel fluoride uptake (EFU) in comparison with the microbiopsy technique. Enamel specimens were exposed to equimolar solutions of fluoride prepared from sodium fluoride (NaF), stannous fluoride (SnF2), or amine fluoride (AmF). EFU was quantified by both techniques on the same specimens. EFU was found to be highest for samples treated with AmF, followed by SnF2 and NaF. Both methods yielded clearly interpretable, highly correlating (r = 0.95) data. ToF-SIMS can be considered a promising alternative to the microbiopsy technique for near-surface EFU assessment.
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Esmalte Dentário , Fluoretos , Espectrometria de Massa de Íon Secundário , Humanos , Aminas , Esmalte Dentário/metabolismo , Fluoretos/administração & dosagem , Projetos Piloto , Fluoreto de Sódio/farmacologia , Fluoreto de Sódio/química , Fluoretos de Estanho/farmacologia , Remineralização Dentária/métodosRESUMO
Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and suboptimal product quality. In addition, these variants may undergo immune reactions, henceforth need to be appropriately controlled to ensure consistent product quality and patient safety. Deamidation of insulin is the most common post-translational modification occurring in insulin and insulin analogues. AsnA21 desamido variant is also the most prominent product variant formed during human insulin manufacturing process and/or during the storage. Often, this deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of purified deamidated material is always being challenging due to highly similar mass, ionic, hydrophobic properties, and high structural similarity of the variant compared to the parent product. Present work demonstrates the simplified and efficient scalable process for generation of AsnA21 deamidated variant in powder form with ~96% purity. The mixed-mode property of anion exchange resin PolyQuat was utilized to purify the deamidated impurity with high recovery. Subsequent reversed-phase high performance liquid chromatography (RP-HPLC) step was introduced for concentration of product in bind elute mode. Elution pool undergone isoelectric precipitation and lyophilisation. The lyophilized product allows users for convenient use of the deamidated impurity for intended purposes. Detailed characterization by Mass spectrometry revealed deamidation is at AsnA21 and further confirmed that, structural and functional characterization as well as the biological activity of isolated variant is equivalent to insulin.
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Insulina/análogos & derivados , Insulina/isolamento & purificação , Processamento de Proteína Pós-Traducional , Cromatografia por Troca Iônica , Cromatografia de Fase Reversa , Liofilização/métodos , Humanos , Insulina/biossíntese , Preparações Farmacêuticas , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Here we report a new α chain variant accidentally discovered during Hb A1c measurement by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) that revealed the presence of a variant α chain with a mass of 15155 Da. However, this hemoglobin (Hb) variant cannot be detected by the first-line methods such as cation exchange high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Sanger sequencing confirmed the presence of a heterozygous missense mutation [HBA2: c.46G>A, codon 15 (GGT>AGT), (GlyâSer)]. The theoretical mass difference (30 Da) due to the substitution of amino acid glycine to serine matched the actual measured mass difference (29 Da). As this is the first report of the mutation, we named it Hb Nanchang after the place of residence of the proband.
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Hemoglobinas Anormais , alfa-Globinas , Códon , Glicina/genética , Hemoglobinas Anormais/análise , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , alfa-Globinas/análise , alfa-Globinas/genéticaRESUMO
Bacteroides pyogenes is part of the normal oral flora of domestic animals. There is one previous report of human infection, with B. pyogenes bacteremia following a cat bite (Madsen 2011). We report seven severe human infections where B. pyogenes was identified by Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDTI-TOF MS), but not by VITEK MS and was misidentified by VITEK ANC card.
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Bacteriemia/microbiologia , Infecções por Bacteroides/microbiologia , Bacteroides/patogenicidade , Mordeduras e Picadas/microbiologia , RNA Ribossômico 16S/genética , Infecção dos Ferimentos/microbiologia , Idoso , Animais , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/patologia , Bacteriemia/cirurgia , Técnicas de Tipagem Bacteriana , Bacteroides/efeitos dos fármacos , Bacteroides/genética , Bacteroides/isolamento & purificação , Infecções por Bacteroides/tratamento farmacológico , Infecções por Bacteroides/patologia , Infecções por Bacteroides/cirurgia , Mordeduras e Picadas/tratamento farmacológico , Mordeduras e Picadas/patologia , Mordeduras e Picadas/cirurgia , Gatos , Criança , Cães , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/patologia , Infecção dos Ferimentos/cirurgiaRESUMO
Preclinical models are essential research tools before novel therapeutic or diagnostic methods can be applied to humans. These range from in vitro cell monocultures to vastly more complex animal models, but clinical translation to humans often fails to deliver significant results. Three-dimensional (3D) organoid systems are being increasingly studied to establish physiologically relevant in vitro platforms in a trade-off between the complexity of the research question and the complexity of practical experimental setups. The sensitivity and precision of analytical tools are yet another limiting factors in what can be investigated, and mass spectrometry (MS) is one of the most powerful analytical techniques available to the scientific community. Its innovative use to spatially resolve biological samples has opened many research avenues in the field of MS imaging (MSI). Here, this work aims to explore the current scientific landscape in the application of MSI on organoids, with an emphasis on their combined potential to facilitate and improve preclinical studies.
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Phosphoinositides serve as essential players in numerous biological activities and are critical for overall cellular function. Due to their complex chemical structures, localization, and low abundance, current challenges in the phosphoinositide field include the accurate measurement and identification of specific variants, particularly those with acyl chains. Researchers are intensively developing innovative techniques and approaches to address these challenges and advance our understanding of the impact of phosphoinositide signaling on cellular biology. This article provides an overview of recent advances in the study of phosphoinositides, including mass spectrometry, lipid biosensors, and real-time activity assays using fluorometric sensors. These methodologies have proven instrumental for a comprehensive exploration of the cellular distribution and dynamics of phosphoinositides and have shed light on the growing significance of these lipids in human health and various pathological processes, including cancer. To illustrate the importance of phosphoinositide signaling in disease, this perspective also highlights the role of a family of lipid kinases named phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), which have recently emerged as exciting therapeutic targets for cancer treatment. The ongoing exploration of phosphoinositide signaling not only deepens our understanding of cellular biology but also holds promise for novel interventions in cancer therapy.
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Lactuca tatarica is a wild species belonging to Asteraceae family omnipresent in Southern Caucasus region including Azerbaijan. Previous studies on the chemical content of some extracts obtained from its different organs have reported the presence of lactone sesquiterpenes, triterpenoids and flavonoids. For the first time, we investigated the volatile composition of L. tatarica aerial parts and seeds by GC/MS technique. The results showed the predominant presence of fatty acids, both saturated and unsaturated. Palmitic acid was prevalent in the aerial parts (up to 89.9%) while linoleic acid (up to 82.6%) was the most abundant component in the seeds. Other minor components were terpene and hydrocarbon derivatives. Some of the detected constituents in L. tatarica have already demonstrated antibacterial, antifungal, anti-inflammatory and antioxidant activity. Therefore, this species could be better studied for its biological properties and considered as a source of active ingredients useful in various fields including the pharmaceutical one.
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Asteraceae , Óleos Voláteis , Componentes Aéreos da Planta/química , Cromatografia Gasosa-Espectrometria de Massas , Terpenos/análise , Antioxidantes/química , Asteraceae/química , Sementes/química , Óleos Voláteis/química , Extratos Vegetais/químicaRESUMO
Overall survival in metastatic lung cancer has been dramatically improved with the use of small molecule kinase inhibitors (SMKIs). Quantification of SMKI in cerebrospinal fluid (CSF) can be used to assess penetration of these drugs into the central nervous system. This paper describes an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantification of the SMKIs alectinib, lorlatinib and osimertinib in human CSF. Alectinib-d8 and dasatinib-d8 were used as internal standards. Aliquots with 25 µL CSF/30% albumin (9:1,v/v) were mixed with 100 µL internal standard solution consisting of 1 ng/mL dasatinib-d8 and alectinib-d8 in acetonitrile. The analytes were separated by an Acquity UPLC® HSS T3 column (2.1 ×150 mm, 1.8 µm), using gradient elution (ammonium formate pH 4.5, acetonitrile) with a flow rate of 0.400 mL/min. All calibration curves were linear for the concentration range from 2.50 to 250 ng/mL. Within-run and between-run precision varied from 0.72% to 11.7%, with accuracy ranging from 95.3% to 113.2%. For all compounds, a high degree of non-specific binding to the vacutainer was observed. This issue could be countered easily by a combination of pre-coating with BSA solution (30%) in phosphate buffer pH 4.2, and immediate sample mixture with BSA solution after collection. To test the clinical applicability, CSF was collected in seven unique patients using alectinib (n = 1), lorlatinib (n = 2), and osimertinib (n = 4). Measured CSF trough concentrations ranged between 3.37 and 116 ng/mL.
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Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Dasatinibe , Lactamas Macrocíclicas , Cromatografia Líquida de Alta Pressão/métodos , Reprodutibilidade dos TestesRESUMO
Listeria monocytogenes is a gram-positive bacillus that causes food poisoning. Listeriosis causes gastrointestinal infections and occasionally leads to fatal bacteremia in older adults. The symptoms of Listeria infections are non-specific and difficult to diagnose. We describe a case of Listeria bacteremia in an 82-year-old Japanese woman who had handled raw venison one month prior to becoming ill, but had not consumed any. No other possible sources of infection were identified. She presented with a fever without any focal symptoms. Computed tomography revealed enteritis with mucosal damage. Blood culture revealed bacteria with gram-positive rod morphology, that were confirmed as L. monocytogenes using mass spectrometry. The patient was treated with intravenous ampicillin and made a full recovery. This case illustrates the virulence of L. monocytogenes, which can cause bacteremia from handling contaminated food, even without consumption.
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Physicochemical tests represent important tools for the analytical control strategy of biotherapeutics. For adenoviral modalities, anion-exchange high performance liquid chromatography (AEX-HPLC) represents an important methodology, as it is able to simultaneously provide information on viral particle concentration, product purity and surface charge in a high-throughput manner. During product development of an adenoviral-based therapeutic, an accelerated stability study was performed and showed changes in each of the AEX-HPLC reportable attributes. These changes also correlated with a decrease in product infectivity prompting a detailed characterization of the impurity and mechanism of the surface charge change. Characterization experiments identified the impurity to be free hexon trimer, suggesting that capsid degradation could be contributing to both the impurity and reduced particle concentration. Additional mass spectrometry characterization identified deamidation of specific hexon residues to be associated with the external surface charge modification observed upon thermal stress conditions. To demonstrate a causal relationship between deamidation and surface charge changes observed by AEX-HPLC, site-directed mutagenesis experiments were performed. Through this effort, it was concluded that deamidation of asparagine 414 was responsible for the surface charge alteration observed in the AEX-HPLC profile but was not associated with the reduction in infectivity. Overall, this manuscript details critical characterization efforts conducted to enable understanding of a pivotal physicochemical test for adenoviral based therapeutics.
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The profound effects of and distress caused by the global COVID-19 pandemic highlighted what has been known in the health sciences a long time ago: that bacteria, fungi, viruses, and parasites continue to present a major threat to human health. Infectious diseases remain the leading cause of death worldwide, with antibiotic resistance increasing exponentially due to a lack of new treatments. In addition to this, many pathogens share the common trait of having the ability to modulate, and escape from, the host immune response. The challenge in medical microbiology is to develop and apply new experimental approaches that allow for the identification of both the microbe and its drug susceptibility profile in a time-sensitive manner, as well as to elucidate their molecular mechanisms of survival and immunomodulation. Over the last three decades, proteomics has contributed to a better understanding of the underlying molecular mechanisms responsible for microbial drug resistance and pathogenicity. Proteomics has gained new momentum as a result of recent advances in mass spectrometry. Indeed, mass spectrometry-based biomedical research has been made possible thanks to technological advances in instrumentation capability and the continuous improvement of sample processing and workflows. For example, high-throughput applications such as SWATH or Trapped ion mobility enable the identification of thousands of proteins in a matter of minutes. This type of rapid, in-depth analysis, combined with other advanced, supportive applications such as data processing and artificial intelligence, presents a unique opportunity to translate knowledge-based findings into measurable impacts like new antimicrobial biomarkers and drug targets. In relation to the Research Topic "Proteomic Approaches to Unravel Mechanisms of Resistance and Immune Evasion of Bacterial Pathogens," this review specifically seeks to highlight the synergies between the powerful fields of modern proteomics and microbiology, as well as bridging translational opportunities from biomedical research to clinical practice.
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BACKGROUND: Within European traditional phytotherapy, extracts from different herbal plants are used for prevention and therapy of uncomplicated urinary tract infections and for flushing out of kidney grits. Besides increased urine flow by slight diuretic effects, also stimulation of Tamm-Horsfall protein (syn. THP, uromodulin) in the distal part of the kidney could explain reduced kidney gravel and anti-virulent activity against uropathogenic E. coli. PURPOSES: Evaluation of THP-inducing activity of extracts from Equisetum arvense, Levisticum officinalis, Ilex paraguariensis, Juniperus communis, Urtica dioica, and Taraxacum officinale by quantification of THP in urine samples after oral application to humans. STUDY DESIGN: 7 days p.o. application of the test intervention to healthy volunteers (n = 10 per intervention group) and analysis of urine samples at day 1 (untreated control values), and days 3, 6 and 8 on THP content by validated ELISA. Antiadhesive activity of urine samples was monitored by flow cytometry using UPEC strain NU14 against human T24 bladder cells. RESULTS: An aqueous extract from E. arvense, fully characterized by a specific LC-MS method, induced THP concentration in urine samples significantly during a 7-day p.o. application up to 300%, related to the untreated controls. Ex vivo investigation of the individual and pooled urine samples with elevated THP concentrations showed good correlation to antiadhesive effects against UPEC NU14 to T24 cells. Urine samples of the Equisetum treated volunteers had no effect on the proliferation and on biofilm formation of UPEC NU14. Silica excretion in the urine samples had no correlation to the respective THP levels. Monitoring of electrolyte content in the urine samples indicat ed diuretic effects of the intervention with Equisetum extract. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 80 compounds from the extract by MS evaluations and 18 compounds by UV detection. This protocol will provide a valuable tool for future quality control of Equisetum extract. CONCLUSION: Aqueous extract from E. arvense significantly stimulates THP secretion in urine samples after 7 days of oral intake and inhibits the interplay between UPEC and bladder host cells. This could explain the therapeutic use of this herbal material for urinary tract infections and kidney gravel. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 82% of all eluted compounds. This protocol will provide a valuable tool for future quality control of Equisetum extract.
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Equisetum , Infecções Urinárias , Escherichia coli Uropatogênica , Diuréticos/farmacologia , Humanos , Extratos Vegetais/química , Infecções Urinárias/tratamento farmacológico , Uromodulina/farmacologia , Uromodulina/uso terapêuticoRESUMO
Quantitative mass-spectrometry-based methods to perform relative and absolute quantification of peptides in the immunopeptidome are growing in popularity as researchers aim to measure the dynamic nature of the peptide major histocompatibility complex repertoire and make copies-per-cell estimations of target antigens of interest. Multiple methods to carry out these experiments have been reported, each with unique advantages and limitations. This article describes existing methods and recent applications, offering guidance for improving quantitative accuracy and selecting an appropriate experimental set-up to maximize data quality and quantity.
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Structurally diverse, specialized lipids are crucial components of microbial membranes and other organelles and play essential roles in ecological functioning. The detection of such lipids in the environment can reveal not only the occurrence of specific microbes but also the physicochemical conditions to which they are adapted to. Traditionally, liquid chromatography coupled with mass spectrometry allowed for the detection of lipids based on chromatographic separation and individual peak identification, resulting in a limited data acquisition and targeting of certain lipid groups. Here, we explored a comprehensive profiling of microbial lipids throughout the water column of a marine euxinic basin (Black Sea) using ultra high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). An information theory framework combined with molecular networking based on the similarity of the mass spectra of lipids enabled us to capture lipidomic diversity and specificity in the environment, identify novel lipids, differentiate microbial sources within a lipid group, and discover potential biomarkers for biogeochemical processes. The workflow presented here allows microbial ecologists and biogeochemists to process quickly and efficiently vast amounts of lipidome data to understand microbial lipids characteristics in ecosystems.
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Cyclin dependent kinase 4/6 inhibitors (CDK4/6i) lead to cell-cycle arrest but also trigger T cell-mediated immunity, which might be mediated by changes in human leukocyte antigen (HLA) ligands. We investigated the effects of CDK4/6i, abemaciclib and palbociclib, on the immunopeptidome at nontoxic levels in breast cancer cell lines by biochemical identification of HLA ligands followed by network analyses. This treatment led to upregulation of HLA and revealed hundreds of induced HLA ligands in breast cancer cell lines. These new ligands were significantly enriched for peptides derived from proteins involved in the "G1/S phase transition of cell cycle" including HLA ligands from CDK4/6, Cyclin D1 and the 26S regulatory proteasomal subunit 4 (PSMC1). Interestingly, peptides from proteins targeted by abemaciclib and palbociclib, were predicted to be the most likely to induce a T cell response. In strong contrast, peptides induced by solely one of the drugs had a lower T cell recognition score compared to the DMSO control suggesting that the observed effect is class dependent. This general hypothesis was exemplified by a peptide from PSMC1 which was among the HLA ligands with highest prediction scores and which elicited a T cell response in healthy donors. Overall, these data demonstrate that CDK4/6i treatment gives rise to drug-induced HLA ligands from G1/S phase transition, that have the highest chance for being recognized by T cells, thus providing evidence that inhibition of a distinct cellular process leads to increased presentation of the involved proteins that may be targeted by immunotherapeutic agents.
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Quinase 6 Dependente de Ciclina , Neoplasias , Quinase 4 Dependente de Ciclina , Humanos , Imunoterapia , Ligantes , Inibidores de Proteínas QuinasesRESUMO
Edible flowers are receiving renewed interest as potential sources of bioactive compounds. The present study aimed to investigate the presence of bioactive compounds and antioxidant activity of some exotic flowers present in Brazil such as Amaranthus hypochondriacus, Tropaeolum majus (red), Tropaeolum majus (orange) and Spilanthes oleracea L. The content of total phenolic compounds, flavonoids, condensed, hydrolysable tannins and antioxidante capacity were determined. The identification and quantification of the phenolic compounds was performed through the UHPLC-QDa-MS system. The compounds p-coumaric acid and ferulic acid were identified and quantified for the first time in all flowers. Tropaeolum majus (red) presented the hightest amounts of total phenolic compounds and hydrolysable tannins. Also, it presented the highest antioxidant capacity for ORAC and FRAP assays. Thus, this study showed the diversity and abudance of natural antioxidants present in edible flowers, which could be explored for application in functional foods and pharmaceuticals.
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Antioxidantes/análise , Flores/química , Compostos Fitoquímicos/análise , Extratos Vegetais/análise , Extratos Vegetais/química , Brasil , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos/análise , Flavonoides/análiseRESUMO
The pathophysiology of irritable bowel syndrome and the detection of biomarkers of specific mechanisms and/or predictors of therapeutic response remain elusive. This roadblock reflects, in large part, the complexity and heterogeneity of the disorder. Recently, there has been growing evidence of a dietary and/or microbiome interaction with the host that may trigger symptoms in a subset of patients. While a number of techniques are available to examine these potential interactions, "omic" approaches such as metabolomics are becoming more widely used. Metabolomics measures hundreds and potentially thousands of known and unknown small molecule chemicals (metabolites) to provide a unique look into mechanisms that underlie symptom generation and potential predictors of therapeutic response. In this issue of the journal, Lee et al use nuclear magnetic resonance (NMR) to demonstrate the value of this approach to study IBS. This review examines the use of metabolomics to better understand IBS, focusing on what has been learned to date, practical and technical considerations, its potential for future research and how the study by Lee et al have contributed to these concepts.
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Síndrome do Intestino Irritável/metabolismo , Metabolômica , Animais , Biologia Computacional , Microbioma Gastrointestinal , Humanos , Síndrome do Intestino Irritável/diagnóstico , Síndrome do Intestino Irritável/microbiologia , Metabolômica/métodosRESUMO
Nowadays, great efforts are made to gain insight into the molecular mechanisms that underlie structural neuronal plasticity. Moreover, the identification of signaling pathways involved in the development of psychiatric disorders aids the screening of possible therapeutic targets. Genetic variations or alterations in GPM6A expression are linked to neurological disorders such as schizophrenia, depression, and Alzheimer's disease. GPM6A encodes the neuronal surface glycoprotein M6a that promotes filopodia/spine, dendrite, and synapse formation by unknown mechanisms. A substantial body of evidence suggests that the extracellular loops of M6a command its function. However, the proteins that associate with them and that modulate neuronal plasticity have not been determined yet. To address this question, we generated a chimera protein that only contains the extracellular loops of M6a and performed a co-immunoprecipitation with rat hippocampus samples followed by TMT/MS. Here, we report 72 proteins, which are good candidates to interact with M6a's extracellular loops and modify its function. Gene ontology (GO) analysis showed that 63% of the potential M6a's interactor proteins belong to the category "synapse," at both sides of the synaptic cleft, "neuron projections" (51%) and "presynapse" (49%). In this sense, we showed that endogenous M6a interacts with piccolo, synaptic vesicle protein 2B, and synapsin 1 in mature cultured hippocampal neurons. Interestingly, about 28% of the proteins left were related to the "myelin sheath" annotation, suggesting that M6a could interact with proteins at the surface of oligodendrocytes. Indeed, we demonstrated the (cis and trans) interaction between M6a and proteolipid protein (PLP) in neuroblastoma N2a cells. Finally, the 72 proteins were subjected to disease-associated genes and variants screening by DisGeNET. Apart from the diseases that have already been associated with M6a, most of the proteins are also involved in "autistic disorder," "epilepsy," and "seizures" increasing the spectrum of disorders in which M6a could play a role. Data are available via ProteomeXchange with identifier PXD017347.
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Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.
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Mapeamento Encefálico/métodos , Encéfalo/metabolismo , Conectoma/métodos , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Aprendizado de Máquina , Mamíferos/fisiologia , Espectrometria de Massas/métodos , Camundongos , Mutação/genéticaRESUMO
Widespread resistance to antimicrobial and cancer therapeutics is evolving in every country worldwide and has a direct impact on global health, agriculture and the economy. The specificity and selectivity of bioactive peptide natural products present a possible stopgap measure to address the ongoing deficit of new therapeutic compounds. PepSAVI-MS (Statistically-guided bioActive Peptides prioritized VIa Mass Spectrometry) is an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. This pipeline was validated via screening of the cyclotide-rich botanical species Viola odorata and identification of the known antimicrobial and anticancer cyclotide cycloviolacin O2. Herein we present and validate novel bioactivities of the anthelmintic V. odorata cyclotide, cycloviolacin O8 (cyO8), including micromolar anticancer activity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines and antifungal activity against the agricultural pathogen Fusarium graminearum. A reduction/alkylation strategy in tandem with PepSAVI-MS analysis also revealed several previously uncharacterized putatively bioactive cyclotides. Downstream implementation of ultraviolet photodissociation (UVPD) tandem mass spectrometry is demonstrated for cyO8 as a method to address traditionally difficult-to-sequence cyclotide species. This work emphasizes the therapeutic and agricultural potential of natural product bioactive peptides and the necessity of developing robust analytical tools to deconvolute nature's complexity.