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Desorption/ionization Time (ionization + time)
Kinds of Desorption/ionization Time Selected AbstractsClassification of cancer types by measuring variants of host response proteins using SELDI serum assaysINTERNATIONAL JOURNAL OF CANCER, Issue 5 2005Eric T. Fung Abstract Protein expression profiling has been increasingly used to discover and characterize biomarkers that can be used for diagnostic, prognostic or therapeutic purposes. Most proteomic studies published to date have identified relatively abundant host response proteins as candidate biomarkers, which are often dismissed because of an apparent lack of specificity. We demonstrate that 2 host response proteins previously identified as candidate markers for early stage ovarian cancer, transthyretin and inter-alpha trypsin inhibitor heavy chain 4 (ITIH4), are posttranslationally modified. These modifications include proteolytic truncation, cysteinylation and glutathionylation. Assays using Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS) may provide a means to confer specificity to these proteins because of their ability to detect and quantitate multiple posttranslationally modified forms of these proteins in a single assay. Quantitative measurements of these modifications using chromatographic and antibody-based ProteinChip® array assays reveal that these posttranslational modifications occur to different extents in different cancers and that multivariate analysis permits the derivation of algorithms to improve the classification of these cancers. We have termed this process host response protein amplification cascade (HRPAC), since the process of synthesis, posttranslational modification and metabolism of host response proteins amplifies the signal of potentially low-abundant biologically active disease markers such as enzymes. © 2005 Wiley-Liss, Inc. [source] PROTEOMIC ANALYSIS IN CARDIOVASCULAR DISEASESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2008C Cieniewski-Bernard SUMMARY 1Cardiovascular diseases are a major cause of morbidity and mortality in western countries. The molecular mechanisms responsible for heart dysfunction are still largely unknown, except in cases of genetic defects or alteration of genes and proteins. 2The publication of genome sequences from humans and other species has demonstrated the complexity of biology, including the finding that one gene does not encode for only one protein but for several, due to mRNA splicing and post-translational modifications. 3Proteomic analysis can provide an overall understanding of changes in the levels of protein expression. Differential proteomics is a powerful tool for improving our understanding of integrated biochemical responses. The main techniques used are two-dimensional electrophoresis (2D-gel) and Surface-Enhanced Laser Desorption/Ionization Time of Flight (SELDI-TOF) to separate proteins associated with mass spectrometry. Bioinformatic tools make it possible to compare protein profiles obtained from diverse biological samples. 4The combination of these approaches has proved to be particularly interesting for studying cardiovascular diseases and thereby improving our understanding of the mechanisms involved and identifying new biochemical factors and biomarkers involved in these diseases. [source] Characterization of natural wax esters by MALDI-TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2009Vladimír Vrkoslav Abstract The applicability of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to the analysis of wax esters (WEs) was investigated. A series of metal salts of 2,5-dihydroxybenzoic acid (DHB) was synthesized and tested as possible matrices. Alkali metal (Li, Na, K, Rb, Cs) and transition metal (Cu, Ag) salts were studied. The matrix properties were evaluated, including solubility in organic solvents, threshold laser power that should be applied for successful desorption/ionization of WEs, the nature of the matrix ions and the mass range occupied by them, and the complexity of the isotope clusters for individual metals. Lithium salt of dihydroxybenzoic acid (LiDHB) performed the best and matrices with purified lithium isotopes (6LiDHB or 7LiDHB) were recommended for WEs. Three sample preparation procedures were compared: (1) mixing the sample and matrix in a glass vial and deposition of the mixture on a MALDI plate (Mix), (2) deposition of sample followed by deposition of matrix (Sa/Ma), and (3) deposition of matrix followed by deposition of sample (Ma/Sa). Morphology of the samples was studied by scanning electron microscopy. The best sample preparation technique was Ma/Sa with the optimum sample to matrix molar ratio 1 : 100. Detection limit was in the low picomolar range. The relative response of WEs decreased with their molecular weight, and minor differences between signals of saturated and monounsaturated WEs were observed. MALDI spectra of WEs showed molecular adducts with lithium [M + Li]+. Fragments observed in postsource decay (PSD) spectra were related to the acidic part of WEs [RCOOH + Li]+ and they were used for structure assignment. MALDI with LiDHB was used for several samples of natural origin, including insect and plant WEs. A good agreement with GC/MS data was achieved. Moreover, MALDI allowed higher WEs to be analyzed, up to 64 carbon atoms in Ginkgo biloba leaves extract. Copyright © 2008 John Wiley & Sons, Ltd. [source] Identification of protein differences between two clinical isolates of Streptococcus mutans by proteomic analysisMOLECULAR ORAL MICROBIOLOGY, Issue 2 2008L. H. Guo Introduction:,Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Different strains of S. mutans may display different virulence mechanisms, so the isolation of the differential proteins is illuminating. Methods:,S. mutans strains 9-1 and 9-2, which both colonized the same oral cavity, were selected after screening for the possession of suspected virulence traits. The soluble cellular proteins were extracted from steady-state planktonic cells of strains 9-1 and 9-2 and were analyzed using high-resolution two-dimensional gel electrophoresis. Then, replicate maps of proteins from the two strains were generated. Proteins expressed only in strain 9-1 or 9-2 were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry, by which peptide mass fingerprints were generated, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Results:, There were 12 proteins only expressed in strain 9-1 and three proteins only expressed in strain 9-2. They were involved in protein biosynthesis, protein folding, cell wall biosynthesis, fatty acid biosynthesis, nucleotide biosynthesis, repair of DNA damage, carbohydrate metabolism, signal transduction, and translation. Conclusion:, The identification of proteins differentially expressed between strains 9-1 and 9-2 provides new information concerning the mechanisms of cariogenesis. [source] The solution structure of the methylated form of the N-terminal 16-kDa domain of Escherichia coli Ada proteinPROTEIN SCIENCE, Issue 3 2006Hiroto Takinowaki N-Ada16k, the N-terminal 16-kDa domain of the Ada protein; meC38 N-Ada16k, the Cys38 methylated form of N-Ada16k; MTase, methyltransferase; HTH, helix-turn-helix; NMR, nuclear magnetic resonance; MALDI-TOF MS, matrix assisted laser desorption/ionization time of flight mass spectrometry; MNU, methylnitrosourea Abstract The N-terminal 16-kDa domain of Escherichia coli Ada protein (N-Ada16k) repairs DNA methyl phosphotriester lesions by an irreversible methyl transfer to its cysteine residue. Upon the methylation, the sequence-specific DNA binding affinity for the promoter region of the alkylation resistance genes is enhanced by 103 -fold. Then, it acts as a transcriptional regulator for the methylation damage. In this paper, we identified the methyl acceptor residue of N-Ada16k and determined the solution structure of the methylated form of N-Ada16k by using NMR and mass spectrometry. The results of a 13C-filtered 1H- 13C HMBC experiment and MALDI-TOF MS and MS/MS experiments clearly showed that the methyl acceptor residue is Cys38. The solution structure revealed that it has two distinct subdomains connected by a flexible linker loop: the methyltransferase (MTase) subdomain with the zinc,thiolate center, and the helical subdomain with a helix-turn-helix motif. Interestingly, there is no potential hydrogen bond donor around Cys38, whereas the other three cysteine residues coordinated to a zinc ion have potential donors. Hence, Cys38 could retain its inherent nucleophilicity and react with a methyl phosphotriester. Furthermore, the structure comparison shows that there is no indication of a remarkable conformational change occurring upon the methylation. This implies that the electrostatic repulsion between the negatively charged DNA and the zinc,thiolate center may avoid the contact between the MTase subdomain and the DNA in the nonmethylated form. Thus, after the Cys38 methylation, the MTase subdomain can bind the cognate DNA because the negative charge of the zinc,thiolate center is reduced. [source] ORIGINAL ARTICLE: Contribution of Interferon-, Receptor 1 Gene Polymorphisms to Pre-Eclampsia in ChinaAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2010Li-Juan Chen Citation Chen L-J, Gao H, Zhou H, Zou L, Zou P. Contribution of interferon-, receptor 1 gene polymorphisms to pre-eclampsia in China. Am J Reprod Immunol 2010; 63: 331,338 Problem, As gene polymorphisms of cytokines receptors have been found to significantly influence cell responses to cytokines, the aim of this study was to test the hypothesis that IFN-, receptor 1 (IFNGR1) gene polymorphisms may contribute to the pathogenesis of pre-eclampsia. Method of study, One hundred and sixty-four pre-eclamptic patients (121 patients with mild pre-eclampsia and 43 patients with severe pre-eclampsia) and 171 controls were included. Polymorphisms of the IFNGR1 gene at positions ,611, ,270, +56 and +95 were genotyped with the matrix-assisted laser desorption/ionization time of flight mass spectrometry. Results, This study showed a positive association between ,56C/C genotype (OR = 1.7; 95% CI = 1.1,2.7) and pre-eclampsia. Although the genotype frequencies (except for ,56C/C) of the two polymorphisms were comparable between cases and controls, higher frequency of the ,611A/,56C haplotype (OR = 1.450; 95% CI = 1.070,1.966) was noticed in patients versus controls. All patients and controls were homozygous for the ,270T/T and +95T/T genotypes. Specifically, the frequency of the ,56C allele (OR = 1.838; 95% CI = 1.127,2.995) was higher among patients with severe pre-eclampsia. Conclusion, The IFNGR1 gene polymorphisms may contribute to the pathogenesis of pre-eclampsia in our population. [source] Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH),APMIS, Issue 11-12 2004BIRGITTA SCHWEICKERT This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2). [source] Purification and characterization of ,-glucosidase in Apis cerana indicaINSECT SCIENCE, Issue 3 2008Chanpen Chanchao Abstract Apis cerana indica foragers were used for the isolation of a full-length ,-glucosidase cDNA, and for purification of the active nascent protein by low salt extraction of bee homogenates, ammonium sulphate precipitation and diethylaminoethyl-cellulose and Superdex 200 chromatographies. The molecular mass of the purified protein was estimated by polyacrylamide gel electrophoresis resolution, and the pH, temperature, incubation, and substrate optima for enzymic activity were determined. Conformation of the purified enzyme as ,-glucosidase was performed by BLAST software homology comparisons between matrix assisted laser desorption ionization time of flight mass spectroscopy analysed partial tryptic peptide digests of the purified protein with the predicted amino acid sequences deduced from the ,-glucosidase cDNA sequence. [source] Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysisJOURNAL OF NEUROCHEMISTRY, Issue 6 2005Marco Morciano Abstract The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl- n -hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate,polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal. [source] Distinct C-terminus of the B subunit of factor XIII in a population-associated major phenotype: the first case of complete allele-specific alternative splicing products in the coagulation and fibrinolytic systemsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009H. IWATA Summary.,Objectives: The purpose of this study was to elucidate the molecular bases of the heterogeneity of the B subunit of coagulation factor XIII (FXIII-B), classified by isoelectric focusing into its three population-associated major phenotypes. Methods and Results: By genetic sequencing and polymerase chain reaction (PCR),restriction fragment length polymorphism analyses, a C-to-G change was identified in intron K for the Asian-associated major phenotype FXIII-B*3. A transcript containing the novel exon XII, was detected by reverse transcription PCR using hepatocyte cell lines with this allele. The exclusive existence of a novel C-terminal peptide in a homozygote of FXIII-B*3 was also detected by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The FXIII-B*3 isoform had a C-terminus 15 residues longer than the other isoforms, containing two additional basic amino acids and one extra acidic amino acid. Accordingly, the C-to-G nucleotide substitution created an efficient splice acceptor AG dinucleotide, which resulted in allele-specific alternative splicing in intron K. When compared with FXIII-B*1, the third major phenotype, FXIII-B*2, had an A-to-G change in exon III, converting His95 to Arg, and a rare phenotype, FXIII-B*4, had an A-to-T change in exon VII, converting Glu368 to Val. Conclusions: We found an extremely rare event of complete allele-specific alternative splicing for FXIII-B. The FXIII-B*3 isoform had a distinct C-terminal peptide, while the FXIII-B*2 and FXIII-B*4 isoforms had His95 to Arg and Glu368 to Val substitutions, respectively, which led to differential isoelectric points of these isoforms. Such variations in the amino acid sequence of FXIII-B may have profound effects on its structure,function relationship, plasma FXIII levels, and disease susceptibility. [source] | ||||||||||||||||