ABSTRACT
Actual research demonstrates that LA-ICP-MS is capable of being used as an imaging tool with cellular resolution. The aim of this investigation was the method development for LA-ICP-MS to extend the versatility to quantitative and multiplexing imaging of single eukaryotic cells. For visualization of individual cells selected, lanthanide-labeled antibodies were optimized for immuno-imaging of single cells with LA-ICP-MS. The molar content of the artificial introduced labels per cell was quantified using self-made nitrocellulose-coated slides for matrix-matched calibration and calculated amounts were in the range of 3.1 to 17.8 atmol per cell. Furthermore, the quantification strategy allows a conversion of 2D intensity profiles based on counts per second (cps) to quantitative 2D profiles representing the molar amount of the artificial introduced elemental probes per pixel for each individual cell. Graphical abstract á .
Subject(s)
Fibroblasts/cytology , Immunohistochemistry/methods , Mass Spectrometry/methods , Single-Cell Analysis/methods , 3T3 Cells , Animals , Antibodies/analysis , Lanthanoid Series Elements/analysis , Mice , Staining and Labeling/methodsABSTRACT
The paper presents a new multi-parametric protein microarray embracing the multi-analyte capabilities of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The combination of high throughput reverse phase protein microarrays with element tagged antibodies and LA-ICP-MS makes it possible to detect and quantify many proteins or biomarkers in multiple samples simultaneously. A proof of concept experiment is performed for the analysis of cytochromes particularly of cytochrome P450 enzymes, which play an important role in the metabolism of xenobiotics such as toxicants and drugs. With the aid of the LA-ICP-MS based multi-parametric reverse phase protein microarray it was possible to analyse 8 cytochromes in 14 different proteomes in one run. The methodology shows excellent detection limits in the lower amol range and a very good linearity of R(2) ≥ 0.9996 which is a prerequisite for the development of further quantification strategies.
Subject(s)
Antibodies/chemistry , Cytochromes/analysis , Laser Therapy/methods , Protein Array Analysis/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Spectrophotometry, Atomic/methods , Animals , Female , Male , Microsomes, Liver/chemistry , Rats , Rats, Sprague-DawleyABSTRACT
Mitochondria fulfill many tissue-specific functions in cell metabolism. We set out to identify differences in the protein composition of mitochondria from five tissues frequently affected by mitochondrial disorders. The proteome of highly purified mitochondria from five mouse organs was separated by high-resolution 2DE. Tissue-specific spots were identified through nano-LC/ESI-MS/MS and quantified by densitometry in ten biological replicates. We identified 87 consistently deviating spots representing 48 proteins. The percentage of variant spots ranged between 4.2% and 6.0%; 21 proteins having tissue-specific isospots. Consistent tissue-specific processing/regulation was seen for carbamoyl-phosphate-synthase, aldehyde-dehydrogenase 2, ATP-synthase α-chain, and isocitrate-dehydrogenase α-subunit. Thirty tissue-specific proteins were associated with mitochondrial disorders in humans. We further identified alcohol-dehydrogenase, catalase, quinone-oxidoreductase, cyclophilin-A, and Upf0317, a potential biotin-carboxyl-carrier protein, which had not been annotated as "mitochondrial" in Gene Ontology or MitoCarta databases. Their targeting to the mitochondria was verified by transfection of full-length GFP-tagged plasmids. Given the high evolutionary conservation of mitochondrial metabolic pathways, these data further annotate the mitochondrial proteome and advance our understanding of the pathophysiology and tissue-specificity of symptoms seen in patients with mitochondrial disorders. The generation of 2D electrophoretic maps of the mitochondrial proteome using tissue specimens in the milligram range facilitates this technique for clinical applications and biomarker research.
Subject(s)
Mitochondria, Heart , Mitochondria, Liver , Mitochondria, Muscle , Mitochondrial Proteins , Animals , Brain/metabolism , Electrophoresis, Gel, Two-Dimensional , Gene Expression , Kidney/metabolism , Mice , Mitochondria, Heart/genetics , Mitochondria, Liver/genetics , Mitochondria, Liver/metabolism , Mitochondria, Muscle/genetics , Mitochondria, Muscle/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Tissue DistributionABSTRACT
UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4's subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the protein's possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.
Subject(s)
Embryo, Mammalian/metabolism , Ion Channels/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Neurons/metabolism , Age Factors , Amino Acid Sequence , Animals , Blotting, Western , Cell Differentiation , Embryo, Mammalian/cytology , Female , Gene Expression Regulation, Developmental , Immunoenzyme Techniques , Immunoglobulin G/immunology , Ion Channels/genetics , Ion Channels/immunology , Mice , Mice, Inbred C57BL , Mitochondrial Proteins/genetics , Mitochondrial Proteins/immunology , Mitochondrial Uncoupling Proteins , Molecular Sequence Data , Neurons/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Subcellular Fractions , Tissue DistributionABSTRACT
Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.
Subject(s)
RNA, Chloroplast/genetics , RNA, Untranslated/genetics , RNA/genetics , Animals , Chloroplasts/genetics , Gene Library , Genome, Plant , Mice , Mitochondria/genetics , RNA/analysis , RNA, Chloroplast/analysis , RNA, Mitochondrial , RNA, Small Nuclear/analysis , RNA, Small Nuclear/genetics , RNA, Untranslated/analysis , Sequence Analysis, DNA , Nicotiana/geneticsABSTRACT
Mitochondrial diseases may be caused by numerous mutations that alter proteins of the respiratory chain and of other metabolic pathways in the mitochondrium. For clinicians this disease group poses a considerable diagnostic challenge due to ambiguous genotype-phenotype relationships. Until now, only 30% of the mitochondriopathies can be diagnosed at the molecular level. We therefore need a new diagnostic tool that offers a wide view on the mitochondrial proteins. Here, we present a method to generate a high-resolution, large-gel two-dimensional gel electrophoretic (2-DE) map of a purified fraction of mitochondrial proteins from Epstein-Barr virus-immortalized lymphoblastoid cell line (LCL). LCLs can be easily obtained from patients and control subjects in a routine clinical setting. They often express the biochemical phenotype and can be cultured to high cell numbers, sufficient to gain enough purified material for 2-DE. In total we identified 166 mitochondrial proteins. Thirteen proteins were earlier not known to be of mitochondrial origin. Thirty-nine proteins were associated with human diseases ranging from respiratory chain enzyme deficiencies to disorders of beta-oxidation and amino acid metabolism. This 2-DE map is intended to be the first step to diagnose mitochondrial diseases at the proteomic level.
