RESUMO
ß-N-methylamino-L-alanine (BMAA) and its isomers, 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)-glycine (AEG), along with microcystins (MCs)-RR, -LR, and -YR (the major MC congeners), are cyanotoxins that can cause detrimental health and environmental impacts during toxic blooms. Currently, there are no reverse-phase (RP) LC-MS/MS methods for the simultaneous detection and quantification of BMAA, its isomers, and the major MCs in a single analysis; therefore, multiple analyses are required to assess the toxic load of a sample. Here, we present a newly developed and validated method for the detection and quantification of BMAA, 2,4-DAB, AEG, MC-LR, MC-RR, and MC-YR using RP LC-MS/MS. Method validation was performed, assessing linearity (r2 > 0.996), accuracy (>90% recovery for spiked samples), precision (7% relative standard deviation), and limits of detection (LODs) and quantification (LOQs) (ranging from 0.13 to 1.38 ng mL-1). The application of this combined cyanotoxin analysis on a culture of Microcystis aeruginosa resulted in the simultaneous detection of 2,4-DAB (0.249 ng mg-1 dry weight (DW)) and MC-YR (4828 ng mg-1 DW). This study provides a unified method for the quantitative analysis of BMAA, its isomers, and three MC congeners in natural environmental samples.
Assuntos
Microcistinas , Espectrometria de Massas em Tandem , Cromatografia Líquida , Toxinas de CianobactériasRESUMO
L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that shares structural similarities with its proteogenic L-proline amino acid counterpart. For this reason, AZE can be misincorporated in place of L-proline, contributing to AZE toxicity. In previous work, we have shown that AZE induces both polarization and apoptosis in BV2 microglial cells. However, it is still unknown if these detrimental effects involve endoplasmic reticulum (ER) stress and whether L-proline co-administration prevents AZE-induced damage to microglia. Here, we investigated the gene expression of ER stress markers in BV2 microglial cells treated with AZE alone (1000 µM), or co-treated with L-proline (50 µM), for 6 or 24 h. AZE reduced cell viability, nitric oxide (NO) secretion and caused a robust activation of the unfolded protein response (UPR) genes (ATF4, ATF6, ERN1, PERK, XBP1, DDIT3, GADD34). These results were confirmed by immunofluorescence in BV2 and primary microglial cultures. AZE also altered the expression of microglial M1 phenotypic markers (increased IL-6, decreased CD206 and TREM2 expression). These effects were almost completely prevented upon L-proline co-administration. Finally, triple/quadrupole mass spectrometry demonstrated a robust increase in AZE-bound proteins after AZE treatment, which was reduced by 84% upon L-proline co-supplementation. This study identified ER stress as a pathogenic mechanism for AZE-induced microglial activation and death, which is reversed by co-administration of L-proline.
Assuntos
Microglia , Prolina , Prolina/farmacologia , Prolina/química , Ácido Azetidinocarboxílico/farmacologia , Ácido Azetidinocarboxílico/química , Aminoácidos , Estresse do Retículo EndoplasmáticoRESUMO
L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its structural similarity with the amino acid L-proline, AZE can evade the editing process during protein assembly in eukaryotic cells and be misincorporated into L-proline-rich proteins, potentially causing protein misfolding and other detrimental effects to cells. In this study, we sought to determine if AZE treatment triggered pro-inflammatory and pro-apoptotic responses in BV2 microglial cells. BV2 microglial cells exposed to AZE at increasing concentrations (0−2000 µM) at 0, 3, 6, 12 and 24 h were assayed for cell viability (MTT) and nitric oxide release (Griess assay). Annexin V-FITC/propidium iodide (PI) staining was used to assess apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and other molecular targets of cell survival response. AZE (at concentrations > 1000 µM) significantly reduced cell viability, increased BAX/Bcl2 ratio and caused cell death. Results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1ß, IL-6, NOS2, CD68 and MHC-2a). Additionally, we found that AZE induced the expression of the extracellular matrix degrading enzyme matrix metalloproteinase 9 (MMP-9) and brain derived neurotrophic factor (BDNF), two critical regulators of microglial motility and structural plasticity. Collectively, these data indicate that AZE-induced toxicity is associated with increased pro-inflammatory activity and reduced survival in BV2 microglia. This evidence may prompt for an increased monitoring of AZE consumption by humans.
RESUMO
ß-N-methylamino L-alanine (BMAA) is a neurotoxin linked to high incidences of neurodegenerative disease. The toxin, along with two of its common isomers, 2,4-diaminobuytric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG), is produced by multiple genera of cyanobacteria worldwide. Whilst there are many reports of locations and species of cyanobacteria associated with the production of BMAA during a bloom, there is a lack of information tracking changes in concentration across a single bloom event. This study aimed to measure the concentrations of BMAA and its isomers through the progression and end of a cyanobacteria bloom event using liquid chromatography-triple quadrupole-mass spectrometry. BMAA was detected in all samples analysed, with a decreasing trend observed as the bloom progressed. BMAA's isomers were also detected in all samples, however, they did not follow the same decreasing pattern. This study highlights the potential for current sampling protocols that measure a single time point as representative of a bloom's overall toxin content to underestimate BMAA concentration during a bloom event.
Assuntos
Diamino Aminoácidos , Cianobactérias , Doenças Neurodegenerativas , Humanos , Diamino Aminoácidos/química , Cianobactérias/química , Cromatografia LíquidaRESUMO
The cyanobacterial non-protein amino acid α-amino-ß-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system xc_ and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA.
Assuntos
Diamino Aminoácidos/efeitos adversos , Autofagia Mediada por Chaperonas , Toxinas de Cianobactérias/efeitos adversos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Neuroblastoma/tratamento farmacológico , Agregados Proteicos/efeitos dos fármacos , Serina/farmacologia , Ubiquitina/metabolismo , Antioxidantes/farmacologia , Diferenciação Celular , Agonistas de Aminoácidos Excitatórios/efeitos adversos , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Células Tumorais CultivadasRESUMO
In order to study the toxicity of the cyanobacterial non-protein amino acids (NPAAs) L-ß-N-methylamino-L-alanine (BMAA) and its structural isomer L-2,4-diaminobutyric acid (DAB) in the forage crop plant alfalfa (Medicago sativa), seedlings were exposed to NPAA-containing media for four days. Root growth was significantly inhibited by both treatments. The content of derivatised free and protein-bound BMAA and DAB in seedlings was then analysed by LC-MS/MS. Both NPAAs were detected in free and protein-bound fractions with higher levels detected in free fractions. Compared to shoots, there was approximately tenfold more BMAA and DAB in alfalfa roots. These results suggest that NPAAs might be taken up into crop plants from contaminated irrigation water and enter the food chain. This may present an exposure pathway for NPAAs in humans.
Assuntos
Diamino Aminoácidos/metabolismo , Aminobutiratos/metabolismo , Produtos Agrícolas/metabolismo , Diamino Aminoácidos/toxicidade , Aminobutiratos/toxicidade , Bioacumulação , Cromatografia Líquida , Produtos Agrícolas/efeitos dos fármacos , Cianobactérias/química , Toxinas de Cianobactérias , Humanos , Isomerismo , Neurotoxinas/análise , Plântula/química , Espectrometria de Massas em TandemRESUMO
In addition to the 20 protein amino acids that are vital to human health, hundreds of naturally occurring amino acids, known as non-proteinogenic amino acids (NPAAs), exist and can enter the human food chain. Some NPAAs are toxic through their ability to mimic protein amino acids and this property is utilised by NPAA-containing plants to inhibit the growth of other plants or kill herbivores. The NPAA L-azetidine-2-carboxylic acid (Aze) enters the food chain through the use of sugar beet (Beta vulgaris) by-products as feed in the livestock industry and may also be found in sugar beet by-product fibre supplements. Aze mimics the protein amino acid L-proline and readily misincorporates into proteins. In light of this, we examined the toxicity of Aze to mammalian cells in vitro. We showed decreased viability in Aze-exposed cells with both apoptotic and necrotic cell death. This was accompanied by alterations in endosomal-lysosomal activity, changes to mitochondrial morphology and a significant decline in mitochondrial function. In summary, the results show that Aze exposure can lead to deleterious effects on human neuron-like cells and highlight the importance of monitoring human Aze consumption via the food chain.
Assuntos
Ácido Azetidinocarboxílico/farmacologia , Morte Celular , Dieta , Mitocôndrias/patologia , Neuroblastoma/patologia , Humanos , Mitocôndrias/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Células Tumorais CultivadasRESUMO
Environmental exposure to the amino acid ß-methylamino-L-alanine (BMAA) was linked to the high incidence of neurodegenerative disease first reported on the island of Guam in the 1940s and has more recently been implicated in an increased incidence of amyotrophic lateral sclerosis (ALS) in parts of the USA. BMAA has been shown to be produced by a range of cyanobacteria and some marine diatoms and dinoflagellates in different parts of the world. BMAA is commonly found with two of its constitutional isomers: 2,4- diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl) glycine (AEG). These isomers are thought to be co-produced by the same organisms that produce BMAA and MS/MS analysis following LC separation can add an additional level of specificity over LC-FL. Although the presence of BMAA and 2,4-DAB in surface scum samples from several sites in Australia has been reported, which Australian cyanobacterial species are capable of BMAA, 2,4-DAB and AEG production remains unknown. The aims of the present studies were to identify some of the cyanobacterial genera or species that can produce BMAA, 2,4-DAB and AEG in freshwater cyanobacteria blooms in eastern Australia. Eleven freshwater sites were sampled and from these, 19 single-species cyanobacterial cultures were established. Amino acids were extracted from cyanobacterial cultures and analysed using liquid chromatography-tandem mass spectrometry. BMAA was detected in 17 of the 19 isolates, 2,4-DAB was detected in all isolates, and AEG was detected in 18 of the 19 isolates, showing the prevalence of these amino acids in Australian freshwater cyanobacteria. Concentrations of all three isomers in Australian cyanobacteria were generally higher than the concentrations reported elsewhere. This study confirmed the presence of BMAA and its isomers in cyanobacteria isolated from eastern Australian freshwater systems, and determined which Australian cyanobacterial genera or species were capable of producing them when cultured under laboratory conditions.
Assuntos
Diamino Aminoácidos/análise , Diamino Aminoácidos/química , Cianobactérias/química , Aminoácidos/análise , Austrália , Cromatografia Líquida , Toxinas de Cianobactérias , Água Doce/microbiologia , Glicina/análise , Glicina/química , Isomerismo , Neurotoxinas/análise , Neurotoxinas/química , Espectrometria de Massas em TandemRESUMO
There is a strong body of evidence linking the non-protein amino acid (NPAA) ß-methylamino-L-alanine (BMAA) to the development of a number of neurodegenerative diseases. BMAA has been found globally, is produced by a number of organisms including cyanobacteria, diatoms, and dinoflagellates; and has been shown to biomagnify through trophic levels. The role of BMAA in neurodegenerative disease is highlighted by its presence in the brains of a number of neurodegenerative disease patients, where it was found in a protein-bound form. We have previously shown that BMAA is bound to cell proteins, and results in the upregulation of the unfolded protein response, an endoplasmic reticulum stress response activated by the presence of misfolded proteins within the cell. Structurally aberrant proteins are features of a number of neurodegenerative diseases, and further investigation of how BMAA interacts with proteins is crucial to our understanding of its toxicity. Here we use radiolabelled BMAA to investigate the interaction and binding of BMAA to eukaryotic and prokaryotic proteins. We found differences in the presence and distribution of protein-bound BMAA between E. coli and neuroblastoma cells, with an increase in binding over time only seen in the eukaryotic cells. We also found that BMAA was unable to bind to pure proteins, or cell lysate in native or denaturing conditions, indicating that biological processing is required for BMAA to bind to proteins.
Assuntos
Diamino Aminoácidos/metabolismo , Encéfalo/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas/metabolismo , Diamino Aminoácidos/toxicidade , Animais , Encéfalo/patologia , Cianobactérias/genética , Cianobactérias/metabolismo , Toxinas de Cianobactérias , Diatomáceas/genética , Diatomáceas/metabolismo , Dinoflagellida/genética , Dinoflagellida/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucarióticas/metabolismo , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Células Procarióticas/metabolismoRESUMO
The 'oxygen paradox' arises from the fact that oxygen, the molecule that aerobic life depends on, threatens its very existence. An oxygen-rich environment provided life on Earth with more efficient bioenergetics and, with it, the challenge of having to deal with a host of oxygen-derived reactive species capable of damaging proteins and other crucial cellular components. In this minireview, we explore recent insights into the metabolism of proteins that have been reversibly or irreversibly damaged by oxygen-derived species. We discuss recent data on the important roles played by the proteasomal and lysosomal systems in the proteolytic degradation of oxidatively damaged proteins and the effects of oxidative damage on the function of the proteolytic pathways themselves. Mitochondria are central to oxygen utilisation in the cell, and their ability to handle oxygen-derived radicals is an important and still emerging area of research. Current knowledge of the proteolytic machinery in the mitochondria, including the ATP-dependent AAA+ proteases and mitochondrial-derived vesicles, is also highlighted in the review. Significant progress is still being made in regard to understanding the mechanisms underlying the detection and degradation of oxidised proteins and how proteolytic pathways interact with each other. Finally, we highlight a few unanswered questions such as the possibility of oxidised amino acids released from oxidised proteins by proteolysis being re-utilised in protein synthesis thus establishing a vicious cycle of oxidation in cells.
Assuntos
Proteínas/metabolismo , Aminoácidos/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The accumulation of oxidised proteins in ageing cells and tissues results from an increase in oxidant damage coupled with impaired degradation of the damaged proteins. Heat Shock Proteins (HSP) and other chaperones are required to recognise damaged proteins and transport them to the lysosomal and proteasomal degradation pathways. How these systems fail in ageing cells is not clear. METHODS: We monitor oxidised protein accumulation, the activity of the proteasome and lysosomal proteases, and HSP levels in MRC-5 fibroblasts throughout their mitotic lifespan. We then use a novel in vitro cell culture model to experimentally generate oxidised proteins in young and old MRC-5 fibroblasts and compare their rates of degradation and changes in the key pathways involved in oxidised protein removal. RESULTS: We show that the activity of the proteasome and some lysosomal enzymes decreases with ageing in MRC-5 cells as do levels of HSP70 but this is not associated with an accumulation of oxidised proteins which only occurs as cells closely approach post-mitotic senescence. Old cells are unable to degrade experimentally generated oxidised proteins as efficiently as young cells. Exposure to mild heat stress however increases the efficiency of oxidised protein degradation by young cells and increases levels of HSP70. CONCLUSIONS: Our results highlight the importance of the HSP/chaperone system in oxidised protein metabolism, particularly in ageing cells. GENERAL SIGNIFICANCE: These data might have implications for the development of therapies for pathologies associated with protein accumulation and suggest that the HSP/chaperone system would be an important target.
Assuntos
Senescência Celular , Fibroblastos/metabolismo , Proteínas/metabolismo , Células Cultivadas , Di-Hidroxifenilalanina/metabolismo , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Oxirredução , Complexo de Endopeptidases do Proteassoma/fisiologiaRESUMO
The cyanobacterial non-protein amino acid (AA) ß-Methylamino-L-alanine (BMAA) is considered to be a neurotoxin. BMAA caused histopathological changes in brains and spinal cords of primates consistent with some of those seen in early motor neuron disease; however, supplementation with L-serine protected against some of those changes. We examined the impact of BMAA on AA concentrations in human neuroblastoma cells in vitro. Cells were treated with 1000 µM BMAA and intracellular free AA concentrations in treated and control cells were compared at six time-points over a 48 h culture period. BMAA had a profound effect on intracellular AA levels at specific time points but in most cases, AA homeostasis was re-established in the cell. The most heavily impacted amino acid was serine which was depleted in BMAA-treated cells from 9 h onwards. Correction of serine depletion could be a factor in the observation that supplementation with L-serine protects against BMAA toxicity in vitro and in vivo. AAs that could potentially be involved in protection against BMAA-induced oxidation such as histidine, tyrosine, and phenylalanine were depleted in cells at later time points.
Assuntos
Diamino Aminoácidos , Neuroblastoma , Animais , Humanos , Aminoácidos , Diamino Aminoácidos/toxicidade , Diamino Aminoácidos/metabolismo , Serina/farmacologia , Neurotoxinas/toxicidadeRESUMO
Cellular deposits of oxidized and aggregated proteins are hallmarks of a variety of age-related disorders, but whether such proteins contribute to pathology is not well understood. We previously reported that oxidized proteins form lipofuscin/ceroid-like bodies with a lysosomal-type distribution and up-regulate the transcription and translation of proteolytic lysosomal enzymes in cultured J774 mouse macrophages. Given the recently identified role of lysosomes in the induction of apoptosis, we have extended our studies to explore a role for oxidized proteins in apoptosis. Oxidized proteins were biosynthetically generated in situ by substituting oxidized analogues for parent amino acids. Apoptosis was measured with Annexin-V/PI (propidium iodide), TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling), MMP (mitochondrial membrane permeabilization), caspase activation and cytochrome c release, and related to lysosomal membrane permeabilization. Synthesized proteins containing the tyrosine oxidation product L-DOPA (L-3,4-dihydroxyphenylalanine) were more potent inducers of apoptosis than proteins containing the phenylalanine oxidation product o-tyrosine. Apoptosis was dependent upon incorporation of oxidized residues, as indicated by complete abrogation in cultures incubated with the non-incorporation control D-DOPA (D-3,4-dihydroxyphenylalanine) or when incorporation was competed out by parent amino acids. The findings of the present study suggest that certain oxidized proteins could play an active role in the progression of age-related disorders by contributing to LMP (lysosomal membrane permeabilization)-initiated apoptosis and may have important implications for the long-term use of L-DOPA as a therapeutic agent in Parkinson's disease.
Assuntos
Apoptose , Levodopa/efeitos adversos , Levodopa/metabolismo , Monócitos/metabolismo , Biossíntese de Proteínas , Tirosina/efeitos adversos , Tirosina/metabolismo , Envelhecimento , Anexina A5/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Ceroide/efeitos adversos , Fragmentação do DNA , Ativação Enzimática , Humanos , Membranas Intracelulares , Lipofuscina/efeitos adversos , Lisossomos , Potencial da Membrana Mitocondrial , Membranas Mitocondriais , Oxirredução , PermeabilidadeRESUMO
Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. ß-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.
Assuntos
Diamino Aminoácidos , Cianobactérias , Síndromes Neurotóxicas , Humanos , Espectrometria de Massas em Tandem/métodos , Diamino Aminoácidos/análise , Diamino Aminoácidos/química , Aminoácidos , Cromatografia Líquida/métodos , Proteínas , Cianobactérias/químicaRESUMO
In the original publication, there was a mistake in Table 2 as published [...].
RESUMO
Proteinopathies are protein misfolding diseases that have an underlying factor that affects the conformation of proteoforms. A factor hypothesised to play a role in these diseases is the incorporation of non-protein amino acids into proteins, with a key example being the therapeutic drug levodopa. The presence of levodopa as a protein constituent has been explored in several studies, but it has not been examined in a global proteomic manner. This paper provides a proof-of-concept method for enzymatically creating levodopa-containing proteins using the enzyme tyrosinase and provides spectral evidence of in vitro incorporation in addition to the induction of the unfolded protein response due to levodopa.
RESUMO
In contrast to mammalian cells, bacteria such as Escherichia coli have been shown to display tolerance towards the neurotoxin ß-methylamino-l-alanine (BMAA) suggesting that these prokaryotes possess a way to metabolise BMAA or its products, resulting in their export, degradation, or detoxification. Single gene deletion mutants of E. coli K-12 with inactivated amino acid biosynthesis pathways were treated with 500 µg/ml BMAA and the resulting growth was monitored. Wild type E. coli and most of the gene deletion mutants displayed unaltered growth in the presence of BMAA over 12 h. Conversely, deletion of genes in the cysteine biosynthesis pathway, cysE, cysK or cysM resulted in a BMAA dose-dependent growth delay in minimal medium. Through further studies of the ΔcysE strain, we observed increased susceptibility to oxidative stress from H2O2 in minimal medium, and disruptions in glutathione levels and oxidation state. The cysteine biosynthesis pathway is therefore linked to the tolerance of BMAA and oxidative stress in E. coli, which potentially represents a mechanism of BMAA detoxification.
Assuntos
Diamino Aminoácidos/farmacologia , Toxinas de Cianobactérias/farmacologia , Cisteína/biossíntese , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Diamino Aminoácidos/metabolismo , Diamino Aminoácidos/toxicidade , Meios de Cultura , Toxinas de Cianobactérias/metabolismo , Toxinas de Cianobactérias/toxicidade , Cisteína Sintase/genética , Tolerância a Medicamentos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Deleção de Genes , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Redes e Vias Metabólicas , Oxirredução , Estresse Oxidativo , Serina O-Acetiltransferase/genéticaRESUMO
LC-MS/MS method development for native amino acid detection can be problematic due to low ionisation efficiencies, in source fragmentation, potential for cluster ion formation and incorrect application of chromatography techniques. This has led to the majority of the scientific community derivatising amino acids for more sensitive analysis. Derivatisation has several benefits including reduced signal-to-noise ratios, more efficient ionisation, and a change in polarity, allowing the use of reverse phase chromatography. However, derivatisation of amino acids can be expensive, requires additional sample preparation steps, is more time consuming and increases sample instability, due to the most derivatised amino acids only be stable for finite amount of time. While showing initial promise, development of reliable hydrophilic interaction liquid chromatography (HILIC) separation methods has presented difficulties for the analyst including irreproducible separation and poor sensitivity. This study aimed to find a means to improve the detection sensitivity of the 20 protein amino acids by HILIC-MS/MS. We describe the use of previously undescribed amino acid-acetonitrile (ACN) adducts to improve detection of 16 out of the 20 amino acids. While all amino acids examined did form an ACN adduct, 4 had low intensity adduct formation compared to their protonated state, 3 of which are classified as basic amino acids. For 15 of the 20 amino acids tested, we used the ACN adduct for both quantification and qualification ions and demonstrated a significant enhancement in signal-to-noise ratio, ranging from 23 to 1762% improvement. Lower LODs, LOQs and lower ranges of linearity were also achieved for these amino acids. The optimised method was applied to a human neuroblastoma cell line (SH-SY5Y) with the potential to be applied to other complex sample types. The improved sensitivity this method offers simplifies sample preparation and reduces the costs of amino acid analysis compared to those methods that rely on derivatisation for sensitivity.
Assuntos
Aminoácidos , Espectrometria de Massas em Tandem , Acetonitrilas , Cromatografia Líquida , Humanos , Interações Hidrofóbicas e HidrofílicasRESUMO
Proteinopathies are diseases caused by factors that affect proteoform conformation. As such, a prevalent hypothesis is that the misincorporation of noncanonical amino acids into a proteoform results in detrimental structures. However, this hypothesis is missing proteomic evidence, specifically the detection of a noncanonical amino acid in a peptide sequence. This review aims to outline the current state of technology that can be used to investigate mistranslations and misincorporations whilst framing the pursuit as Misincorporation Proteomics (MiP). The current availability of technologies explored herein is mass spectrometry, sample enrichment/preparation, data analysis techniques, and the hyphenation of approaches. While many of these technologies show potential, our review reveals a need for further development and refinement of approaches is still required.
RESUMO
Elevated levels of oxidized proteins are reported in diseased tissue from age-related pathologies such as atherosclerosis, neurodegenerative disorders, and cataract. Unlike the precise mechanisms that exist for the repair of nucleic acids, lipids, and carbohydrates, the primary pathway for the repair of oxidized proteins is complete catabolism to their constitutive amino acids. This process can be inefficient as is evidenced by their accumulation. It is generally considered that damaged proteins are degraded by the proteasome; however, this is only true for mildly oxidized proteins, because substrates must be unfolded to enter the narrow catalytic core. Rather, evidence suggests that moderately or heavily oxidized proteins are endocytosed and enter the endosomal/lysosomal system, indicating co-operation between the proteasomes and the lysosomes. Heavily modified substrates are incompletely degraded and accumulate within the lysosomal compartments resulting in the formation of lipofuscin-like, autofluorescent aggregates. Accumulation eventually results in impaired turnover of large organelles such as proteasomes and mitochondria, lysosomal destablization, leakage of proteases into the cytosol and apoptosis. In this review, we summarize reports published since our last assessments of the field of oxidized protein degradation including a role for modified proteins in the induction of apoptosis.