RESUMO
Proteomics is one of the most significant methodologies to better understand the molecular pathways involved in diseases and to improve their diagnosis, treatment and follow-up. The investigation of the proteome of complex organisms is challenging from an analytical point of view, because of the large number of proteins present in a wide range of concentrations. In this study, nanofluidic chromatography, using a micropillar array column, was coupled to drift-tube ion mobility and time-of-flight mass spectrometry to identify as many proteins as possible in a protein digest standard of HeLa cells. Several chromatographic parameters were optimized. The high interest of drift-tube ion mobility to increase the number of identifications and to separate isobaric coeluting peptides was demonstrated. Multiplexed drift-tube ion mobility spectrometry was also investigated, to increase the sensitivity in proteomics studies. This innovative proteomics platform will be useful for analyzing patient samples to better understand unresolved disorders.
Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Células HeLa , Humanos , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Proteoma , Proteômica/métodosRESUMO
The discovery of new antigens specific to multiple myeloma that could be targeted by novel immunotherapeutic approaches is currently of great interest. To this end, it is important to increase the number of proteins identified in the sample by combining different separation strategies. A capillary zone electrophoresis (CZE) method, coupled with drift tube ion mobility (DTIMS) and quadrupole time-of-flight mass spectrometry (QTOF), was developed for antigen discovery using the human myeloma cell line LP-1. This method was first optimized to obtain a maximum number of identifications. Then, its performance in terms of uniqueness of identifications was compared to data acquired by a microfluidic reverse phase liquid chromatography (RPLC) method. The orthogonality of these two approaches and the physicochemical properties of the entities identified by CZE and RPLC were evaluated. In addition, the contribution of DTIMS to CZE was investigated in terms of orthogonality as well as the ability to provide unique information. In conclusion, we believe that the combination of CZE-DTIMS-QTOF and microfluidic RPLC provides unique information in the context of antigen discovery.
Assuntos
Cromatografia de Fase Reversa , Mieloma Múltiplo , Humanos , Espectrometria de Massas em Tandem/métodos , Microfluídica , Linhagem Celular Tumoral , Eletroforese Capilar/métodosRESUMO
In recent years, lipidomics have been widely developed to try to better understand many diseases or physical conditions. In this study, the aim was to evaluate the possibility to conduct reliable lipidomic studies using hemaPEN® microsampling devices. Targeted lipidomic analysis was applied to investigate the impact of a short and intense physical activity on lipids blood concentration. HemaPEN® microsampling device was used to easily collect several samples directly on an athletics track. This device allows the accurate collection of four blood samples (2.74 µL each) in a non-invasive way and without any specific skills. In this study, nineteen healthy volunteers aged from 19 to 27 were included. Participants ran 400 m warm-up and 1600 m as fast as possible. Blood samples were collected at five different time points. One sample was collected before the exercise, two during the physical activity and two after. An extraction process as well as an ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method were optimized to follow-up 11 compounds in these small volumes of blood. Blood concentration of five out of the eleven targeted analytes were significantly influenced by the physical exercise. Blood concentration of arachidonic acid, sphingosine and lactic acid were significantly increased after exercise, while concentration of 14:0 lysophosphatidylcholine and 18:1 lysophosphatidylcholine were significantly decreased.
Assuntos
Lipidômica , Espectrometria de Massas em Tandem , Humanos , Idoso , Cromatografia Líquida de Alta Pressão/métodos , Esforço Físico , Lisofosfatidilcolinas , AtletasRESUMO
Hydrophilic interaction liquid chromatography (HILIC) coupled to drift tube ion mobility spectrometry (DTIMS) was used to separate diastereomers of five-unit oligonucleotides containing 0, 1, 2 or 3 phosphorothioate (PS) linkages. Multiplexed DTIMS (where ions are pulsed into the drift tube according to a pre-encoded sequence) and post-acquisition processing using an innovative demultiplexing tool were investigated. The electric field inside the drift tube was optimized to achieve the highest resolving power. The entrance voltage providing the best two-peak resolution was -1000V with 3-bit multiplexing. Under optimized conditions, the eight diastereomers of an oligonucleotide with three PS linkages (5'-TC∗G∗T∗G-3') could be separated unambiguously. Indeed, those diastereomers differed in their collision cross section (CCS) values. The minimal CCS values difference between two adjacent diastereomers was 0.9% with maximal RSD on CCS values of 0.3%. The use of multiplexed ion mobility and the novel high-resolution demultiplexing tool represents a real breakthrough for resolution enhancement of diastereomers in linear DTIMS.
Assuntos
Espectrometria de Mobilidade Iônica , Oligonucleotídeos , Cromatografia Líquida , Íons , Espectrometria de MassasRESUMO
Monitoring approaches and technical improvements are key factors to improve a sportsman's health, training, and recovery after an injury. In this study, a targeted metabolomics approach using microsampling with hemaPEN® was developed to measure changes in blood concentrations of nine amino acids and four organic acids before, during, and after exercise. The aim of this research project was to investigate if a reliable monitoring of metabolite levels during sports activity can be achieved by collecting one drop of whole blood at different time points. A hemaPEN device is an easy-to-use and noninvasive microsampling technique designed to collect four accurate and precise blood volumes simultaneously (10.96 µl). Twenty healthy volunteers between 19 and 30 years of age were included in this study. Physical activity consisted in running as fast as possible 1,600 m after 400 m warm-up. One drop of blood was collected at five time points: before exercise, after 800-m running, after 1,600 m, and 30 min and 60 min after finishing the exercise. The influence of physical activity on metabolite levels was evaluated using two ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) methods. Analytical performance criteria such as metabolite stability, method precision, trueness, and accuracy were found to be satisfactory. Expected significant metabolic changes were identified for lactic acid, main TCA cycle intermediates, and some amino acids (e.g., creatinine, choline, and taurine). This preliminary study performed on a small cohort demonstrated a high interest of using microsampling for fluxomics analysis, not only to collect quickly and easily biological samples during sports events but also because it is much easier to store and to process the samples than classical plasma/serum samples obtained by venipuncture. The present results open new avenue for fluxomics analysis in the context of health care.
RESUMO
Ion mobility (IM) mass spectrometry allows conducting data independent acquisition (DIA) where all ions entering the instrument are fragmented based on their drift time. In this work, DIA operational parameters were first optimized using a design of experiments. The optimization of data treatment involved a smoothing algorithm of the IM dimension, which increased the number of identified peptides. Then, classical DDA and IM-based DIA were compared injecting increasing amounts of a complex proteome digest (E. coli). Results revealed that compared to DDA, DIA allowed to identify from 2 to 3.3 times more proteins, depending on the injected quantity. To evaluate proteome coverage, endogenous proteins in E. coli cells were sorted by abundance deciles. A large majority of the proteins uniquely observed in DDA were part of the 10% most abundant protein groups. Interestingly, owing to the absence of ion-picking algorithm, DIA allowed to identify proteins coming from a broader concentration range therefore greatly improving proteome coverage. Furthermore, ion mobility separation improved coverage by separating co-eluting peptides. Physicochemical properties of peptides uniquely detected by DIA or DDA were also compared using supervised and unsupervised multivariate analysis. As a result, peptides having a higher mass and being relatively hydrophobic were significantly more identified in DIA. Finally, semi-quantitative performance of both methods was investigated and proved to be comparable, except that DIA demonstrated a better sensitivity than DDA. As a conclusion, we demonstrated in this study that both acquisition modes provide complementary information about the proteome under investigation.
Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Algoritmos , Animais , Bovinos , Escherichia coli/química , Proteínas de Escherichia coli/análise , Peptídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Volumetric absorptive microsampling (VAMS) enables the collection of small and accurate quantities of biological fluids. Therefore, this sampling technique is of great interest for volume-limited samples or serial collection of samples. In this study, we examined the potential of VAMS for targeted mass spectrometry (MS)-based metabolomics. The targeted analysis of 36 major metabolites from only 10⯵L of whole blood was optimized. A design of experiments was carried out to maximize the extraction of metabolites. Moreover, critical steps in sample preparation and sample analysis were studied and characterized, such as the addition of internal standards to tips of VAMS devices before sample collection. A reversed-phase UHPLC-MS/MS method was used to analyze organic acids, whereas hydrophilic interaction chromatography (HILIC)-MS/MS was selected for the determination of amino acids. Overall, the optimum extraction solvent was acetonitrile-water in a proportion of 60:40 (v/v), providing good recoveries and resulting in the detection of all target metabolites in whole blood with good repeatability (less than 15% RSD on peak area). Furthermore, the stability of the analytes in dried whole blood, which is of critical importance in metabolomics studies, was investigated. The amino and organic acids were stable for at least 4 days when stored at room temperature. This is in contrast to the instability of these compounds in wet blood, thereby showing the great potential of VAMS in metabolomics studies.