RESUMO
This review covers the results of the application of mass spectrometric (MS) techniques to study the diversity of beta-amyloid (Aß) peptides in human samples. Since Aß is an important hallmark of Alzheimer's disease (AD), which is a socially significant neurodegenerative disorder of the elderly worldwide, analysis of its endogenous variations is of particular importance for elucidating the pathogenesis of AD, predicting increased risks of the disease onset, and developing effective therapy. MS approaches have no alternative for the study of complex samples, including a wide variety of Aß proteoforms, differing in length and modifications. Approaches based on matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography with electrospray ionization tandem MS are most common in Aß studies. However, Aß forms with isomerized and/or racemized Asp and Ser residues require the use of special methods for separation and extra sensitive and selective methods for detection. Overall, this review summarizes current knowledge of Aß species found in human brain, cerebrospinal fluid, and blood plasma; focuses on application of different MS approaches for Aß studies; and considers the potential of MS techniques for further studies of Aß-peptides.
RESUMO
Intrauterine growth restriction (IUGR) remains a significant concern in modern obstetrics, linked to high neonatal health problems and even death, as well as childhood disability, affecting adult quality of life. The role of maternal and fetus adaptation during adverse pregnancy is still not completely understood. This study aimed to investigate the disturbance in biological processes associated with isolated IUGR via blood plasma proteomics. The levels of 125 maternal plasma proteins were quantified by liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) with corresponding stable isotope-labeled peptide standards (SIS). Thirteen potential markers of IUGR (Gelsolin, Alpha-2-macroglobulin, Apolipoprotein A-IV, Apolipoprotein B-100, Apolipoprotein(a), Adiponectin, Complement C5, Apolipoprotein D, Alpha-1B-glycoprotein, Serum albumin, Fibronectin, Glutathione peroxidase 3, Lipopolysaccharide-binding protein) were found to be inter-connected in a protein-protein network. These proteins are involved in plasma lipoprotein assembly, remodeling, and clearance; lipid metabolism, especially cholesterol and phospholipids; hemostasis, including platelet degranulation; and immune system regulation. Additionally, 18 proteins were specific to a particular type of IUGR (early or late). Distinct patterns in the coagulation and fibrinolysis systems were observed between isolated early- and late-onset IUGR. Our findings highlight the complex interplay of immune and coagulation factors in IUGR and the differences between early- and late-onset IUGR and other placenta-related conditions like PE. Understanding these mechanisms is crucial for developing targeted interventions and improving outcomes for pregnancies affected by IUGR.
Assuntos
Retardo do Crescimento Fetal , Proteômica , Gravidez , Adulto , Recém-Nascido , Feminino , Humanos , Criança , Retardo do Crescimento Fetal/metabolismo , Qualidade de Vida , Feto/metabolismo , Placenta/metabolismoRESUMO
Bleomycin, which is widely used as an antitumor agent, possesses serious adverse effects such as pulmonary toxicity. Local nanoaerosol deposition for lung cancer treatment is a promising alternative to drug delivery to lung lesions. The aim of this work is to test the hypothesis that bleomycin nanoaerosol can be effectively used to treat multiple lung metastases. To obtain bleomycin nanoaerosol, an aerosol generator based on electrospray of a solution of a nonvolatile substance with gas-phase neutralization of charged aerosol particles was used. Lung metastases in murine Lewis lung carcinoma and B16 melanoma animal models were counted. The effect of inhaled bleomycin nanoparticles on the number and volume of metastases, as well as pulmonary side effects, was investigated. Using a mouse exposure chamber, the dose-dependent effect of inhaled bleomycin on tumor volume was evaluated in comparison with intraperitoneal administration. Bleomycin nanoaerosol reduced the volume of metastases and produced a higher antitumor effect at much lower doses. It has been established that long-term exposure to nanoaerosol with a low dose of bleomycin is capable of suppressing cancer cell growth. The treatment was well tolerated. In the lungs, minor changes were found in the form of focal-diffuse infiltration of the lung parenchyma.
Assuntos
Carcinoma , Neoplasias Pulmonares , Animais , Camundongos , Bleomicina/toxicidade , Aerossóis e Gotículas Respiratórios , Pulmão , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Carcinoma/patologiaRESUMO
Glomerulopathies with nephrotic syndrome that are resistant to therapy often progress to end-stage chronic kidney disease (CKD) and require timely and accurate diagnosis. Targeted quantitative urine proteome analysis by mass spectrometry (MS) with multiple-reaction monitoring (MRM) is a promising tool for early CKD diagnostics that could replace the invasive biopsy procedure. However, there are few studies regarding the development of highly multiplexed MRM assays for urine proteome analysis, and the two MRM assays for urine proteomics described so far demonstrate very low consistency. Thus, the further development of targeted urine proteome assays for CKD is actual task. Herein, a BAK270 MRM assay previously validated for blood plasma protein analysis was adapted for urine-targeted proteomics. Because proteinuria associated with renal impairment is usually associated with an increased diversity of plasma proteins being present in urine, the use of this panel was appropriate. Another advantage of the BAK270 MRM assay is that it includes 35 potential CKD markers described previously. Targeted LC-MRM MS analysis was performed for 69 urine samples from 46 CKD patients and 23 healthy controls, revealing 138 proteins that were found in ≥2/3 of the samples from at least one of the groups. The results obtained confirm 31 previously proposed CKD markers. Combination of MRM analysis with machine learning for data processing was performed. As a result, a highly accurate classifier was developed (AUC = 0.99) that enables distinguishing between mild and severe glomerulopathies based on the assessment of only three urine proteins (GPX3, PLMN, and A1AT or SHBG).
Assuntos
Falência Renal Crônica , Insuficiência Renal Crônica , Humanos , Proteoma , Espectrometria de Massas/métodos , Proteinúria/diagnóstico , Proteínas Sanguíneas , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/urina , BiomarcadoresRESUMO
Mass spectrometry (MS)-based quantitative proteomic methods have become some of the major tools for protein biomarker discovery and validation. The recently developed parallel reaction monitoring-parallel accumulation-serial fragmentation (prm-PASEF) approach on a Bruker timsTOF Pro mass spectrometer allows the addition of ion mobility as a new dimension to LC-MS-based proteomics and increases proteome coverage at a reduced analysis time. In this study, a prm-PASEF approach was used for the multiplexed absolute quantitation of proteins in human plasma using isotope-labeled peptide standards for 125 plasma proteins, over a broad (104-106) dynamic range. Optimization of LC and MS parameters, such as accumulation time and collision energy, resulted in improved sensitivity for more than half of the targets (73 out of 125 peptides) by increasing the signal-to-noise ratio by a factor of up to 10. Overall, 41 peptides showed up to a 2-fold increase in sensitivity, 25 peptides showed up to a 5-fold increase in sensitivity, and 7 peptides showed up to a 10-fold increase in sensitivity. Implementation of the prm-PASEF method allowed absolute protein quantitation (down to 1.13 fmol) in human plasma samples. A comparison of the concentration values of plasma proteins determined by MRM on a QTRAP instrument and by prm-PASEF on a timsTOF Pro revealed an excellent correlation (R2 = 0.97) with a slope of close to 1 (0.99), demonstrating that prm-PASEF is well suited for "absolute" quantitative proteomics.
Assuntos
Proteoma , Proteômica , Proteínas Sanguíneas , Humanos , Espectrometria de Massas , Peptídeos/análise , Proteômica/métodosRESUMO
Alzheimer's disease (AD) is the most common socially significant neurodegenerative pathology, which currently affects more than 30 million elderly people worldwide. Since the number of patients grows every year and may exceed 115 million by 2050, and due to the lack of effective therapies, early prediction of AD remains a global challenge, solution of which can contribute to the timely appointment of a preventive therapy in order to avoid irreversible changes in the brain. To date, clinical assays for the markers of amyloidosis in cerebrospinal fluid (CSF) have been developed, which, in conjunction with the brain MRI and PET studies, are used either to confirm the diagnosis based on obligate clinical criteria or to predict the risk of AD developing at the stage of mild cognitive impairment (MCI). However, the problem of predicting AD at the asymptomatic stage remains unresolved. In this regard, the search for new protein markers and studies of proteomic changes in CSF and blood plasma are of particular interest and may consequentially identify particular pathways involved in the pathogenesis of AD. Studies of specific proteomic changes in blood plasma deserve special attention and are of increasing interest due to the much less invasive method of sample collection as compared to CSF, which is important when choosing the object for large-scale screening. This review briefly summarizes the current knowledge on proteomic markers of AD and considers the prospects of developing reliable methods for early identification of AD risk factors based on the proteomic profile.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Idoso , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Biomarcadores , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Disfunção Cognitiva/diagnóstico , Humanos , Proteômica , Proteínas tauRESUMO
Chronic liver diseases affect more than 1 billion people worldwide and represent one of the main public health issues. Nonalcoholic fatty liver disease (NAFLD) accounts for the majority of mortal cases, while there is no currently approved therapeutics for its treatment. One of the prospective approaches to NAFLD therapy is to use a mixture of natural compounds. They showed effectiveness in alleviating NAFLD-related conditions including steatosis, fibrosis, etc. However, understanding the mechanism of action of such mixtures is important for their rational application. In this work, we propose a new dereplication workflow for deciphering the mechanism of action of the lignin-derived natural compound mixture. The workflow combines the analysis of molecular components with high-resolution mass spectrometry, selective chemical tagging and deuterium labeling, liver tissue penetration examination, assessment of biological activity in vitro, and computational chemistry tools used to generate putative structural candidates. Molecular docking was used to propose the potential mechanism of action of these structures, which was assessed by a proteomic experiment.
Assuntos
Aprendizado Profundo , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Lignina/farmacologia , Polifenóis/farmacologia , Polifenóis/uso terapêutico , Polifenóis/análise , Proteômica , Simulação de Acoplamento Molecular , Espectrometria de MassasRESUMO
Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.
Assuntos
Glomerulosclerose Segmentar e Focal , Nefrose Lipoide , Humanos , Nefrose Lipoide/diagnóstico , Nefrose Lipoide/metabolismo , Nefrose Lipoide/patologia , Glomerulosclerose Segmentar e Focal/metabolismo , Cistatina C/metabolismo , Proteômica , Gelsolina/metabolismo , Proteoma/metabolismo , Hemopexina/metabolismo , Vitronectina/metabolismo , Fator I do Complemento/metabolismo , Vitamina A/metabolismo , Biomarcadores , Esteroides , Vitamina DRESUMO
Early recognition of the risk of Alzheimer's disease (AD) onset is a global challenge that requires the development of reliable and affordable screening methods for wide-scale application. Proteomic studies of blood plasma are of particular relevance; however, the currently proposed differentiating markers are poorly consistent. The targeted quantitative multiple reaction monitoring (MRM) assay of the reported candidate biomarkers (CBs) can contribute to the creation of a consistent marker panel. An MRM-MS analysis of 149 nondepleted EDTA-plasma samples (MHRC, Russia) of patients with AD (n = 47), mild cognitive impairment (MCI, n = 36), vascular dementia (n = 8), frontotemporal dementia (n = 15), and an elderly control group (n = 43) was performed using the BAK 125 kit (MRM Proteomics Inc., Canada). Statistical analysis revealed a significant decrease in the levels of afamin, apolipoprotein E, biotinidase, and serum paraoxonase/arylesterase 1 associated with AD. Different training algorithms for machine learning were performed to identify the protein panels and build corresponding classifiers for the AD prognosis. Machine learning revealed 31 proteins that are important for AD differentiation and mostly include reported earlier CBs. The best-performing classifiers reached 80% accuracy, 79.4% sensitivity and 83.6% specificity and were able to assess the risk of developing AD over the next 3 years for patients with MCI. Overall, this study demonstrates the high potential of the MRM approach combined with machine learning to confirm the significance of previously identified CBs and to propose consistent protein marker panels.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Idoso , Doença de Alzheimer/diagnóstico , Biomarcadores , Proteínas Sanguíneas , Disfunção Cognitiva/diagnóstico , Humanos , Aprendizado de Máquina , Espectrometria de Massas , ProteômicaRESUMO
Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Técnicas Bacteriológicas/instrumentação , Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Membrana Transportadoras/genética , Técnicas Bacteriológicas/métodos , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Glicólise , Glioxilatos/metabolismo , Fenômenos Magnéticos , Oxigênio/metabolismo , Aldeído Pirúvico/metabolismo , Voo Espacial , Ausência de PesoRESUMO
In this work, we demonstrate a new approach for interactively assessing hyperspectral data spatial structures for heterogeneity using mass spectrometry imaging. This approach is based on the visualization of the cosine distance as the similarity levels between mass spectra of a chosen region and the rest of the image (sample). The applicability of the method is demonstrated on a set of mass spectrometry images of frontal mouse brain slices. Selection of the reference pixel of the mass spectrometric image and a further view of the corresponding cosine distance map helps to prepare supporting vectors for further analysis, select features, and carry out biological interpretation of different tissues in the mass spectrometry context with or without histological annotation. Visual inspection of the similarity maps reveals the spatial distribution of features in tissue samples, which can serve as the molecular histological annotation of a slide.
Assuntos
Diagnóstico por Imagem , Testes Diagnósticos de Rotina , Animais , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Alzheimer's disease (AD) is the leading cause of dementia among the elderly. Neuropathologically, AD is characterized by the deposition of a 39- to 42-amino acid long ß-amyloid (Aß) peptide in the form of senile plaques. Several post-translational modifications (PTMs) in the N-terminal domain have been shown to increase the aggregation and cytotoxicity of Aß, and specific Aß proteoforms (e.g., Aß with isomerized D7 (isoD7-Aß)) are abundant in the senile plaques of AD patients. Animal models are indispensable tools for the study of disease pathogenesis, as well as preclinical testing. In the presented work, the accumulation dynamics of Aß proteoforms in the brain of one of the most widely used amyloid-based mouse models (the 5xFAD line) was monitored. Mass spectrometry (MS) approaches, based on ion mobility separation and the characteristic fragment ion formation, were applied. The results indicated a gradual increase in the Aß fraction of isoD7-Aß, starting from approximately 8% at 7 months to approximately 30% by 23 months of age. Other specific PTMs, in particular, pyroglutamylation, deamidation, and oxidation, as well as phosphorylation, were also monitored. The results for mice of different ages demonstrated that the accumulation of Aß proteoforms correlate with the formation of Aß deposits. Although the mouse model cannot be a complete analogue of the processes occurring in the human brain in AD, and several of the observed parameters differ significantly from human values supposedly due to the limited lifespan of the model animals, this dynamic study provides evidence on at least one of the possible mechanisms that can trigger amyloidosis in AD, i.e., the hypothesis on the relationship between the accumulation of isoD7-Aß and the progression of AD-like pathology.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Fosforilação/fisiologia , Placa Amiloide/metabolismoRESUMO
Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.
Assuntos
Ferritinas/genética , Poríferos/genética , Animais , Sequência Conservada , Ferritinas/química , Ferritinas/metabolismo , Ferro/metabolismo , Redes e Vias Metabólicas/genética , Modelos Moleculares , Filogenia , Poríferos/classificação , Poríferos/metabolismo , Domínios Proteicos/genética , Análise de Sequência de DNA , Transcriptoma/fisiologiaRESUMO
The detection of viral RNA by polymerase chain reaction (PCR) is currently the main diagnostic tool for COVID-19 ( Eurosurveillance 2019, 25 (3), 1). The PCR-based test, however, shows limited sensitivity, especially in the early and late stages of disease development ( Nature 2020, 581, 465-469; J. Formosan Med. Assoc. 2020, 119 (6) 1123), and is relatively time-consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemic conditions. Mass spectrometry is one of such possibilities. We have developed a mass-spectrometry-based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs based on the detection of the viral nucleocapsid N protein. Our approach shows confident identification of the N protein in patient samples, even those with the lowest viral loads, and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and the addition of isopropanol and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.
Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Espectrometria de Massas/métodos , Nasofaringe/virologia , Proteínas do Nucleocapsídeo/análise , Pneumonia Viral/diagnóstico , Betacoronavirus/química , COVID-19 , Teste para COVID-19 , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Mucosa Nasal/virologia , Pandemias , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fosfoproteínas , Pneumonia Viral/virologia , Proteômica , SARS-CoV-2 , Carga ViralRESUMO
Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization. Graphical Abstract.
Assuntos
Ácido Aspártico/análise , Modelos Teóricos , Peptídeos/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Isomerismo , Espectrometria de Massas/métodos , Probabilidade , Reprodutibilidade dos TestesRESUMO
The study of urinary peptidome is an important area of research, which concerns the characterization of endogenous peptides, as well as the identification of biomarkers for a wide range of socially significant diseases. First of all, this relates to renal and genitourinary pathologies and/or pathologies associated with proteinuria, such as kidney diseases, bladder, prostate and ovarian cancers, diabetic nephropathy, and pre-eclampsia. Unlike proteins, peptides do not require proteolytic hydrolysis, can be analyzed in their native form and can provide certain information about occurring (patho)physiological processes. Mass spectrometry (MS)-based approaches are the most unbiased and sensitive instruments with high multiplexing capacity and provided most of the current information about endogenous urine peptides. However, despite the large number of urine peptidomic studies, there are certain issues related to the insufficient comparability of their results due to the lack of consistent approaches to their interpretation. Also the development of a custom project-specific protein library for endogenous peptides search and identification is another important point that should be noted in the context of high-throughput peptidomic analysis. Here we propose the custom-specific urinary protein database and the grouping of endogenous urinary peptides with overlapping sequences as useful tools, which can facilitate the acquisition and analysis of LC-MS peptidomic data, as well as the comparison of results of different studies, which should facilitate their more efficient further application.
Assuntos
Espectrometria de Massa com Cromatografia Líquida , Espectrometria de Massas em Tandem , Humanos , Masculino , Feminino , Gravidez , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida , Proteínas , Peptídeos/metabolismo , Proteômica/métodosAssuntos
Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/sangue , Espectrometria de Massas/métodos , Técnicas de Diagnóstico Molecular/métodos , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/química , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Humanos , Espectrometria de Massas/tendências , Técnicas de Diagnóstico Molecular/tendênciasRESUMO
Metastasis is a serious and often life-threatening condition, representing the leading cause of death among women with breast cancer (BC). Although the current clinical classification of BC is well-established, the addition of minimally invasive laboratory tests based on peripheral blood biomarkers that reflect pathological changes in the body is of utmost importance. In the current study, the serum proteome and lipidome profiles for 50 BC patients with (25) and without (25) metastasis were studied. Targeted proteomic analysis for concertation measurements of 125 proteins in the serum was performed via liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) using the BAK 125 kit (MRM Proteomics Inc., Victoria, BC, Canada). Untargeted label-free lipidomic analysis was performed using liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS), in both positive and negative ion modes. Finally, 87 serum proteins and 295 lipids were quantified and showed a moderate correlation with tumor grade, histological and biological subtypes, and the number of lymph node metastases. Two highly accurate classifiers that enabled distinguishing between metastatic and non-metastatic BC were developed based on proteomic (accuracy 90%) and lipidomic (accuracy 80%) features. The best classifier (91% sensitivity, 89% specificity, AUC = 0.92) for BC metastasis diagnostics was based on logistic regression and the serum levels of 11 proteins: alpha-2-macroglobulin, coagulation factor XII, adiponectin, leucine-rich alpha-2-glycoprotein, alpha-2-HS-glycoprotein, Ig mu chain C region, apolipoprotein C-IV, carbonic anhydrase 1, apolipoprotein A-II, apolipoprotein C-II and alpha-1-acid glycoprotein 1.
RESUMO
Despite the differences in the clinical manifestations of major obstetric syndromes, such as preeclampsia (PE) and intrauterine growth restriction (IUGR), their pathogenesis is based on the dysregulation of proliferation, differentiation, and invasion of cytotrophoblast cells that occur in the developing placenta, decidual endometrium, and myometrial parts of the spiral arteries. To understand the similarities and differences in the molecular mechanisms of PE and IUGR, samples of the placental bed and placental tissue were analyzed using protein mass spectrometry and the deep sequencing of small RNAs, followed by validation of the data obtained by quantitative RT-PCR in real time. A comparison of the transcriptome and proteomic profiles in the samples made it possible to conclude that the main changes in the molecular profile in IUGR occur in the placental bed, in contrast to PE, in which the majority of molecular changes occurs in the placenta. In placental bed samples, significant changes in the ratio of miRNA and its potential target gene expression levels were revealed, which were unique for IUGR (miR-30c-5p/VIM, miR-28-3p/VIM, miR-1-3p/ANXA2, miR-30c-5p/FBN1; miR-15b-5p/MYL6), unique for PE (miR-185-3p/FLNA), common for IUGR and PE (miR-30c-5p/YWHAZ and miR-654-3p/FGA), but all associated with abnormality in the hemostatic and vascular systems as well as with an inflammatory process at the fetalâmaternal interface.
RESUMO
C-reactive protein, cystatin C, myoglobin, and D-dimer represent the inflammatory or thromboembolic status of the patient and play important roles in early diagnostics of acute myocardial infarction. Each protein can indicate some health problems, but their simultaneous detection can be crucial for differential diagnostics. The express analysis of these proteins in a small drop of plasma was developed using magnetic beads. The suggested method is based on immunomagnetic extraction of the target analyte from plasma samples and its simultaneous labelling by fluorescent dye. Reaction time was optimized for quantification of cardiac biomarkers in the spike solutions and human plasma samples. In this paper, we developed a one-protein detection technique for each cardiac biomarker and united it to a four-protein facility using an automatic platform. The proposed technique requires only 17 µL of the human plasma and takes 14 min for four-protein measuring. The suggested technique covers concentration difference by more than two orders of magnitude and demonstrates analytical applicability by measurements of human plasma samples of 16 volunteers.