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RATIONALE: Despite decades of implementation, the selection of optimal sample preparation conditions for matrix-assisted laser desorption/ionization (MALDI) imaging is still ambiguous due to the lack of a universal and comprehensive evaluation methodology. Thus, numerous experiments with different matrix application conditions accompany a translation of the method to novel sample types and matrices. METHODS: Mouse brain tissues were covered with 9-aminoacridine through sublimation, followed by recrystallization in vapors of 5% (v/v) methanol solution in water. The samples were analyzed by MALDI time-of-flight mass spectrometry, and the efficiency of lipid and small-molecule ionization was evaluated with different metrics. RESULTS: We first investigate the dependency of matrix density and recrystallization conditions on the thickness of an analyte-empty matrix layer to roughly evaluate the laser shot number required to obtain an intense signal with minimal noise. Then, we introduce metrics for the analysis of small imaging datasets (small sample regions) of model samples based on median quantity of peaks in spectra (medQP) and weighted median signal-to-noise ratio (wmSNR). The evaluation of small regions and taking median values for metrics help overcome the sample heterogeneity and allow for the simultaneous comparison of different acquisition parameters. CONCLUSIONS: Here, we propose a methodology based on gradual laser ablation of small regions of sample and further implementation of weighted signal-to-noise ratio to assess various matrix application conditions. The proposed approach helps reduce the number of test samples required to determine optimal sample preparation conditions and improve the overall quality of images.
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Rapid and reliable methods for detecting tumor margins are crucial for neuro-oncology. Several mass spectrometry-based methods have been recently proposed to address this problem. Inline Cartridge Extraction (ICE) demonstrates the potential for clinical application, based on ex-vivo analysis of dissected tissues, but requires time-consuming steps to avoid cross-contamination. In this work, a method of incorporating a disposable electrospray emitter into the ICE cartridge by PEEK sleeves melting is developed. It reduces total analysis time and improves throughput. The proposed setup also improves the robustness of the ICE molecular profiling as demonstrated with human glial tumor samples in that stability and reproducibility of the spectra were increased.
Assuntos
Espectrometria de Massas por Ionização por Electrospray , Humanos , Espectrometria de Massas por Ionização por Electrospray/métodos , Reprodutibilidade dos TestesRESUMO
Ex-vivo molecular profiling has recently emerged as a promising method for intraoperative tissue identification, especially in neurosurgery. The short-term storage of resected samples at room temperature is proposed to have negligible influence on the lipid molecular profiles. However, a detailed investigation of short-term molecular profile stability is required to implement molecular profiling in a clinic. This study evaluates the effect of storage media, temperature, and washing solution to determine conditions that provide stable and reproducible molecular profiles, with the help of ambient ionization mass spectrometry using rat cerebral cortex as model brain tissue samples. Utilizing normal saline for sample storage and washing media shows a positive effect on the reproducibility of the spectra; however, the refrigeration shows a negligible effect on the spectral similarity. Thus, it was demonstrated that up to hour-long storage in normal saline, even at room temperature, ensures the acquisition of representative molecular profiles using ambient ionization mass spectrometry.
Assuntos
Encéfalo , Solução Salina , Animais , Lipídeos/análise , Espectrometria de Massas , Ratos , Reprodutibilidade dos TestesRESUMO
Ambient ionization mass spectrometry has become one of the most promising approaches for rapid and high-throughput screening of small molecules in complex biological matrices for emergency medicine, forensics, and food and agriculture applications. The simple procedures for sample collection and ionization without additional pretreatment are vital in these fields. Many efforts have been devoted to modifying various ambient ionization techniques to simplify the procedures and improve the robustness and sensitivity of the methods. Here, we demonstrate the implementation of rigid spherical sampler probes to improve the robustness of touch spray ionization mass spectrometry. The sphericity of the probes increases the stability of the cone-jet mode of electrospray, reduces the requirements for fine positioning of a sampler in the ion source, and decreases the possibility of corona discharge occurrence. The utilization of spherical sampler probes allows fast, non-invasive sampling, followed by rapid analysis for various drugs of different chemical classes in complex biological matrices, such as the whole blood or sebum collected from the skin surface. The linearity of the analytical signal response from drug concentration confirms the possibility of creating a simple semiquantitative method for small molecules monitoring using spherical sampler probes.
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Ensaios de Triagem em Larga Escala/métodos , Preparações Farmacêuticas/análise , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , HumanosRESUMO
INTRODUCTION: Lung cancer remains the most prevalent cause of cancer mortality worldwide mainly due to insufficient availability of early screening methods for wide-scale application. Exhaled breath condensate (EBC) is currently considered as one of the promising targets for early screening and is particularly attractive due to its absolutely noninvasive collection and possibility for long-term frozen storage. EBC proteome analysis can provide valuable information about the (patho)physiological changes in the respiratory system and may help to identify in time a high risk of lung cancer. Mass spectrometry (MS) profiling of EBC proteome seems to have no alternative in obtaining the most extensive data and characteristic marker panels for screening. AREAS COVERED: This special report summarizes the data of several proteomic studies of EBC in normal and lung cancer (from 2012 to 2021, PubMed), focuses on the possible reasons for the significant discrepancy in the results, and discusses some aspects for special attention in further studies. EXPERT OPINION: The significant discrepancy in the results of various studies primarily highlights the need to create standardized protocols for the collection and preparation of EBC for proteomic analysis. The application of quantitative and targeted LC-MS/MS based approaches seems to be the most promising in further EBC proteomic studies.
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Neoplasias Pulmonares , Proteoma , Biomarcadores , Biomarcadores Tumorais , Testes Respiratórios , Cromatografia Líquida , Humanos , Pulmão , Neoplasias Pulmonares/diagnóstico , Proteômica , Espectrometria de Massas em TandemRESUMO
Data normalization is an essential part of a large-scale untargeted mass spectrometry metabolomics analysis. Autoscaling, Pareto scaling, range scaling, and level scaling methods for liquid chromatography-mass spectrometry data processing were compared with the most common normalization methods, including quantile normalization, probabilistic quotient normalization, and variance stabilizing normalization. These methods were tested on eight datasets from various clinical studies. The efficiency of the data normalization was assessed by the distance between clusters corresponding to batches and the distance between clusters corresponding to clinical groups in the space of principal components, as well as by the number of features with a pairwise statistically significant difference between the batches and the number of features with a pairwise statistically significant difference between clinical groups. Autoscaling demonstrated the most effective reduction in interbatch variation and can be preferable to probabilistic quotient or quantile normalization in liquid chromatography-mass spectrometry data.
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Tumor cell percentage (TCP) is an essential characteristic of biopsy samples that directly affects the sensitivity of molecular testing in clinical practice. Apart from clarifying diagnoses, rapid evaluation of TCP combined with various neuronavigation systems can be used to support decision making in neurosurgery. It is known that ambient mass spectrometry makes it possible to rapidly distinguish healthy from malignant tissues. In connection with this, here we demonstrate the possibility of using non-imaging ambient mass spectrometry to evaluate TCP in glial tumor tissues with a high degree of confidence. Molecular profiles of histologically annotated human glioblastoma tissue samples were obtained using the inline cartridge extraction ambient mass spectrometry approach. XGBoost regressors were trained to evaluate tumor cell percentage. Using cross-validation, it was estimated that the TCP was determined by the regressors with a precision of approximately 90% using only low-resolution data. This result demonstrates that ambient mass spectrometry provides an accurate method todetermine TCP in dissected tissues even without implementing mass spectrometry imaging. The application of such techniques offers the possibility to automate routine tissue screening and TCP evaluation to boost the throughput of pathology laboratories. Rapid estimation of tumor cell percentage during neurosurgery.
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Neoplasias Encefálicas/patologia , Encéfalo/patologia , Glioblastoma/patologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Biópsia , Encéfalo/cirurgia , Neoplasias Encefálicas/cirurgia , Glioblastoma/cirurgia , HumanosRESUMO
Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry can be used for rapid quantitation of peptides with various post-translational modifications (PTM), even if they do not shift the mass of the native peptide. Previously, it was shown that MALDI-TOF MS can be used for quantitation of isoD7 beta-amyloid 1-42 peptide. On the basis of the differences in the collision-induced dissociation fragmentation pattern of native Aß, isoD7 Aß, isoD23 Aß, and isoD7_23 peptide (a di-isomerized peptide with both isomerization of D7 and D23 residues), we developed a MALDI-TOF-based method for simultaneous quantitation of all of these isoforms. Using multivariate regression for analysis of fragment MS data, the method allows the determination of the molar fractions of all of these isoforms with up to 16% error for mixtures with 2 pmol total amount of the beta-amyloid peptide.
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Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fracionamento Químico/métodos , Isomerismo , Análise Multivariada , Dinâmica não LinearRESUMO
The study of protein misfolding and post-translational processing abnormalities is a promising diagnostic approach for socially significant pathologies associated with the accumulation of abnormal forms of proteins. Recently, it was shown that amyloid-like aggregates can be observed in the urine of pregnant women with preeclampsia, which is the most severe hypertensive complication that can lead to fateful outcomes. The protein composition of urine aggregates may clarify the molecular mechanisms underlying the pathology and has not yet been studied in detail. Using a proteomic approach based on high-resolution mass spectrometry, we studied the protein composition of amyloid-like structures that aggregate in the presence of Congo red azo-dye in the urine of pregnant women with preeclampsia. Fragments of ß-sheets of α-1-antitrypsin, complement 3, haptoglobin, ceruloplasmin, and trypstatin were identified as most likely targets for Congo red binding.
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Amiloide/urina , Espectrometria de Massas/métodos , Pré-Eclâmpsia/urina , Proteoma/análise , Amiloide/química , Vermelho Congo , Feminino , Humanos , Pré-Eclâmpsia/metabolismo , Gravidez , Proteoma/química , Proteômica/métodosRESUMO
The development of perspective diagnostic techniques in medicine requires efficient high-throughput biological sample analysis methods. Here, we present an inline cartridge extraction that facilitates the screening rate of mass spectrometry shotgun lipidomic analysis of tissue samples. We illustrate the method by its application to tumor tissue identification in neurosurgery. In perspective, this high-performance method provides new possibilities for the investigation of cancer pathogenesis and metabolic disorders.
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Neoplasias Encefálicas/metabolismo , Espectrometria de Massas , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Feminino , Humanos , MasculinoRESUMO
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.
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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
Humic substances (HS) are complex natural mixtures comprising a large variety of compounds produced during decomposition of decaying biomass. The molecular composition of HS is extremely diverse as it was demonstrated with the use of high resolution mass spectrometry. The building blocks of HS are mostly represented by plant-derived biomolecules (lignins, lipids, tannins, carbohydrates, etc.). As a result, HS show a wide spectrum of biological activity. Despite that, HS remain a 'biological activity black-box' due to unknown structures of constituents responsible for the interaction with molecular targets. In this study, we investigated the antiviral activity of eight HS fractions isolated from peat and coal, as well as of two synthetic humic-like materials. We determined molecular compositions of the corresponding samples using ultra-high resolution Fourier-transform ion cyclotron resonance mass-spectrometry (FTICR MS). Inhibitory activity of HS was studied with respect to reproduction of tick-borne encephalitis virus (TBEV), which is a representative of Flavivirus genus, and to a panel of enteroviruses (EVs). The samples of natural HS inhibited TBEV reproduction already at a concentration of 1 µg/mL, but they did not inhibit reproduction of EVs. We found that the total relative intensity of FTICR MS formulae within elemental composition range commonly attributed to flavonoid-like structures is correlating with the activity of the samples. In order to surmise on possible active structural components of HS, we mined formulae within FTICR MS assignments in the ChEMBL database. Out of 6502 formulae within FTICR MS assignments, 3852 were found in ChEMBL. There were more than 71 thousand compounds related to these formulae in ChEMBL. To support chemical relevance of these compounds to natural HS we applied the previously developed approach of selective isotopic exchange coupled to FTICR MS to obtain structural information on the individual components of HS. This enabled to propose compounds from ChEMBL, which corroborated the labeling data. The obtained results provide the first insight onto the possible structures, which comprise antiviral components of HS and, respectively, can be used for further disclosure of antiviral activity mechanism of HS.
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Antivirais/química , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Substâncias Húmicas/análise , Solo/química , Antivirais/análise , Antivirais/farmacologia , Biomassa , Carvão Mineral , Mineração de Dados , Bases de Dados de Compostos Químicos , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Humanos , Reprodução/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Cervicovaginal fluid (CVF) is a valuable source of clinical information about the female reproductive tract in both nonpregnant and pregnant women. The aim of this study is to specify the CVF proteome at different stages of cervix neoplastic transformation by label-free quantitation approach based on liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The proteome composition of CVF from 40 women of reproductive age with human papillomavirus (HPV)-associated cervix neoplastic transformation (low-grade squamous intraepithelial lesion [LSIL], high-grade squamous intraepithelial lesion [HSIL], and CANCER) was investigated. Hierarchical clustering and principal component analysis (PCA) of the proteomic data obtained by a label-free quantitation approach show the distribution of the sample set between four major clusters (no intraepithelial lesion or malignancy [NILM], LSIL, HSIL and CANCER) depending on the form of cervical lesion. Multisample ANOVA with subsequent Welch's t test resulted in 117 that changed significantly across the four clinical stages, including 27 proteins significantly changed in cervical cancer. Some of them were indicated as promising biomarkers previously (ACTN4, VTN, ANXA1, CAP1, ANXA2, and MUC5B). CVF proteomic data from the discovery stage were analyzed by the partial least squares-discriminant analysis (PLS-DA) method to build a statistical model, allowing to differentiate severe dysplasia (HSIL and CANCER) from the mild/normal stage (NILM and LSIL), and receiver operating characteristic (ROC) area under the curve (AUC) were obtained on an independent set of 33 samples. The sensitivity of the model was 77%, and the specificity was 94%; AUC was equal to 0.87. CVF proteome proved to be reflect the stage of cervical epithelium neoplastic process.
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Líquidos Corporais/metabolismo , Transformação Celular Neoplásica/metabolismo , Colo do Útero/metabolismo , Proteoma/análise , Neoplasias do Colo do Útero/diagnóstico , Vagina/metabolismo , Adulto , Biomarcadores/análise , Transformação Celular Neoplásica/patologia , Colo do Útero/patologia , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Papillomaviridae/fisiologia , Infecções por Papillomavirus/metabolismo , Gravidez , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Vagina/patologia , Esfregaço VaginalRESUMO
BACKGROUND: The conditions of space flight have a significant effect on the physiological processes in the human body, yet the molecular mechanisms driving physiological changes remain unknown. METHODS: Blood samples of 18 Russian cosmonauts who had conducted long-duration missions to the International Space Station were collected 30 days before launch and on the first and seventh days after landing. RESULTS: A panel of 125 proteins in the blood plasma was quantitated by a well-established and highly regarded targeted mass spectrometry approach. This method involves the monitoring of multiple reactions in conjunction with stable isotope-labeled standards at the University of Victoria - Genome BC Proteomics Centre. CONCLUSIONS: Reduction of circulating plasma volume during space flight and activation of fluid retention at the final stage of the flight affect the changes in plasma protein concentrations present in the first days after landing. Using an ANOVA approach, it was revealed that only 1 protein (S100A9) reliably responded to space flight conditions. This protein plays an important role in the functioning of the endothelium and can serve as a marker for activation of inflammatory reactions. Concentrations of the proteins of complement, coagulation cascades, and acute phase reactants increase in the blood of cosmonauts as measured the first day after landing. Most of these proteins' concentrations continue to increase by the 7th day after space flight. Similar dynamics are observed for proteases and their inhibitors. Thus, there is a shift in proteolytic blood systems, which is necessary for the restoration of muscle tissue and maintenance of oncotic homeostasis.
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Proteínas Sanguíneas/metabolismo , Proteômica/métodos , Voo Espacial , Adulto , Proteínas Sanguíneas/genética , Calgranulina B/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Marcação por Isótopo , Masculino , Espectrometria de Massas , Pessoa de Meia-IdadeAssuntos
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
Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (â¼350â¯nm excitation; â¼450â¯nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.
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Amiloide/química , Insulina/química , Muramidase/química , Agregados Proteicos , Aminoácidos/química , Animais , Benzotiazóis/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Cinética , Oxirredução , Espectrometria de FluorescênciaRESUMO
Urine is a sample of choice for noninvasive biomarkers search because it is easily available in large amounts and its molecular composition provides information on processes in the organism. The high potential of urine peptidomics has been demonstrated for clinical purpose. Several mass spectrometry based approaches have been successfully applied for urine peptidome analysis and potential biomarkers search. Summarizing literature data and our own experience we developed a protocol for comprehensive urine peptidome analysis. The technology includes several stages and consists of urine sample preparation by size exclusion chromatography and identification of featured peptides by nano-HPLC coupled to Fourier transform ion cyclotron resonance mass spectrometry, semiquantitative and statistical data analysis.
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Cromatografia Líquida de Alta Pressão/métodos , Ciclotrons/instrumentação , Fragmentos de Peptídeos/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Urinálise/métodos , HumanosRESUMO
Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder. Amyloid-ß (Aß) aggregation is likely to be the major cause of AD. In contrast to humans and other mammals, that share the same Aß sequence, rats and mice are invulnerable to AD-like neurodegenerative pathologies, and Aß of these rodents (ratAß) has three amino acid substitutions in the metal-binding domain 1-16 (MBD). Angiotensin-converting enzyme (ACE) cleaves Aß-derived peptide substrates, however, there are contradictions concerning the localization of the cleavage sites within Aß and the roles of each of the two ACE catalytically active domains in the hydrolysis. In the current study by using mass spectrometry and molecular modelling we have tested a set of peptides corresponding to MBDs of Aß and ratAß to get insights on the interactions between ACE and these Aß species. It has been shown that the N-domain of ACE (N-ACE) acts as an arginine specific endopeptidase on the Aß and ratAß MBDs with C-amidated termini, thus assuming that full-length Aß and ratAß can be hydrolyzed by N-ACE in the same endopeptidase mode. Taken together with the recent data on the molecular mechanism of zinc-dependent oligomerization of Aß, our results suggest a modulating role of N-ACE in AD pathogenesis.
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Peptídeos beta-Amiloides/metabolismo , Arginina/metabolismo , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Domínios e Motivos de Interação entre Proteínas , Serina Endopeptidases/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Humanos , Hidrólise , Espectrometria de Massas , Modelos Moleculares , Conformação Molecular , Proteólise , Ratos , Especificidade por SubstratoRESUMO
A novel atmospheric pressure thermal ionization (APTI) ion source was developed for the analysis of liquid samples. The feasibility of the ion source was demonstrated on peptides using two configurations-assisted by hot wire or hot surface. Microalloyed molybdenum, used as a thermal ion- emitter, notably facilitated the formation of multiply-charged ions, but fragmentation products were still observed. Peptide fragmentation accompanying thermal ionization can be used for peptide identification. The described method is promising for studies of biological samples with minimal pre-treatment.
Assuntos
Pressão Atmosférica , Calefação/instrumentação , Mapeamento de Peptídeos/métodos , Peptídeos/análise , Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A serious problem during intensive care and nursing of premature infants is the invasiveness of many examination methods. Urine is an excellent source of potential biomarkers due to the safety of the collection procedure. The purpose of this study was to determine the features specific for the urine proteome of preterm newborns and their changes under respiratory pathologies of infectious and non-infectious origin. The urine proteome of 37 preterm neonates with respiratory diseases and 10 full-term newborns as a control group were investigated using the LC-MS/MS method. The total number of identified proteins and unique peptides was 813 and 3672 respectively. In order to further specify the defined infant-specific dataset these proteins were compared with urine proteome of healthy adults (11 men and 11 pregnant women) resulting in 94 proteins found only in infants. Pairwise analysis performed for label-free proteomic data revealed 36 proteins which reliably distinguished newborns with respiratory disorders of infectious genesis from those with non-infectious pathologies, including: proteins involved in cell adhesion (CDH-2,-5,-11, NCAM1, TRY1, DSG2), metabolism (LAMP1, AGRN, TPP1, GPX3, APOD, CUBN, IDH1), regulation of enzymatic activity (SERPINA4, VASN, GAPDH), inflammatory and stress response (CD55, CD 93, NGAL, HP, TNFR, LCN2, AGT, S100P, SERPINA1/C1/B1/F1).