RESUMO
Myocardial infarction is the most common cause of death worldwide. An understanding of the alterations in protein pathways is needed in order to develop strategies that minimize myocardial damage. To identify the protein signature of cardiac ischemia/reperfusion (I/R) injury in rats, we combined, for the first time, protein matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and label-free proteomics on the same tissue section placed on a conductive slide. Wistar rats were subjected to I/R surgery and sacrificed after 24 h. Protein MALDI-MSI data revealed ischemia specific regions, and distinct profiles for the infarct core and border. Firstly, the infarct core, compared to histologically unaffected tissue, showed a significant downregulation of cardiac biomarkers, while an upregulation was seen for coagulation and immune response proteins. Interestingly, within the infarct tissue, alterations in the cytoskeleton reorganization and inflammation were found. This work demonstrates that a single tissue section can be used for protein-based spatial-omics, combining MALDI-MSI and label-free proteomics. Our workflow offers a new methodology to investigate the mechanisms of cardiac I/R injury at the protein level for new strategies to minimize damage after MI.
Assuntos
Doença da Artéria Coronariana , Infarto do Miocárdio , Traumatismo por Reperfusão , Animais , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Infarto do Miocárdio/patologia , ReperfusãoRESUMO
Determination of plasma vitamin B12 (B12) is a frequently requested laboratory analysis, mainly employed to establish B12 deficiency. However, an increased level of B12 is a common unexpected finding that may be related to an increased concentration of one of the B12 binding proteins, haptocorrin or transcobalamin. This paper describes the extensive laboratory evaluation of a patient with an elevated level of plasma B12 with various well-established assays. Initial studies suggested the presence of a macromolecule consisting of haptocorrin bound B12. Specific determinations of the B12-binding proteins revealed normal amounts of haptocorrin but a markedly increase in both total and B12 saturated transcobalamin (holo-TC). The results are in accord with the presence of macro-transcobalamin. These experiments reveal that determination of the nature of the B12-macromolecules is troublesome due to differences in assays applied to measure these proteins. In addition, this publication creates awareness of macro-holo-TC as a cause of an unexplained increased B12 level.
Assuntos
Transcobalaminas , Deficiência de Vitamina B 12 , Humanos , Transcobalaminas/análise , Vitamina B 12RESUMO
Mass spectrometry imaging (MSI) can analyze the spatial distribution of hundreds of different molecules directly from tissue sections usually placed on conductive glass slides to provide conductivity on the sample surface. Additional experiments are often required for molecular identification using consecutive sections on membrane slides compatible with laser capture microdissection (LMD). In this work, we demonstrate for the first time the use of a single conductive slide for both matrix-assisted laser desorption ionization (MALDI)-MSI and direct proteomics. In this workflow, regions of interest can be directly ablated with LMD while preserving protein integrity. These results offer an alternative for MSI-based multimodal spatial-omics.
Assuntos
Proteômica/instrumentação , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida/métodos , Microdissecção e Captura a Laser , Espectrometria de Massas em TandemAssuntos
Biomarcadores/sangue , Exercício Físico , Corrida/fisiologia , Troponina T/sangue , HumanosRESUMO
Background Cardiac troponin T ( cTnT ) is seen in many other conditions besides myocardial infarction, and recent studies demonstrated distinct forms of cTnT . At present, the in vivo formation of these different cTnT forms is incompletely understood. We therefore performed a study on the composition of cTnT during the course of myocardial infarction, including coronary venous system sampling, close to its site of release. Methods and Results Baseline samples were obtained from multiple coronary venous system locations, and a peripheral artery and vein in 71 non- ST -segment-elevation myocardial infarction patients. Additionally, peripheral blood was drawn at 6- and 12-hours postcatheterization. cTnT concentrations were measured using the high-sensitivity- cTnT immunoassay. The cTnT composition was determined via gel filtration chromatography and Western blotting in an early and late presenting patient. High-sensitivity - cTnT concentrations were 28% higher in the coronary venous system than peripherally (n=71, P<0.001). Coronary venous system samples demonstrated cT n T-I-C complex, free intact cTnT , and 29 kD a and 15 to 18 kD a cTnT fragments, all in higher concentrations than in simultaneously obtained peripheral samples. While cT n T-I-C complex proportionally decreased, and disappeared over time, 15 to 18 kD a cTnT fragments increased. Moreover, cT n T-I-C complex was more prominent in the early than in the late presenting patient. Conclusions This explorative study in non- ST -segment-elevation myocardial infarction shows that cTnT is released from cardiomyocytes as a combination of cT n T-I-C complex, free intact cTnT , and multiple cTnT fragments indicating intracellular cTnT degradation. Over time, the cT n T-I-C complex disappeared because of in vivo degradation. These insights might serve as a stepping stone toward a high-sensitivity- cTnT immunoassay more specific for myocardial infarction.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Vasos Coronários , Infarto do Miocárdio sem Supradesnível do Segmento ST/sangue , Troponina T/sangue , Idoso , Western Blotting , Cromatografia em Gel , Seio Coronário , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , Isoformas de Proteínas/sangue , Troponina C/sangue , Troponina I/sangue , Troponina T/metabolismoRESUMO
Mass spectrometry imaging (MSI) is a widely established technology; however, in the cardiovascular research field, its use is still emerging. The technique has the advantage of analyzing multiple molecules without prior knowledge while maintaining the relation with tissue morphology. Particularly, MALDI-based approaches have been applied to obtain in-depth knowledge of cardiac (dys)function. Here, we discuss the different aspects of the MSI protocols, from sample handling to instrumentation used in cardiovascular research, and critically evaluate these methods. The trend towards structural lipid analysis, identification, and "top-down" protein MSI shows the potential for implementation in (pre)clinical research and complementing the diagnostic tests. Moreover, new insights into disease progression are expected and thereby contribute to the understanding of underlying mechanisms related to cardiovascular diseases.