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Absolute Quantitation (absolute + quantitation)
Selected AbstractsQuantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewageLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2004M.A. Laverick Abstract Aims:, Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. Methods and Results:, Noroviruses in environmental water samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler® and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3,-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. Conclusions:, Sewage was found to contain up to 1·8 × 106 norovirus cDNA copies per 100 ml and effluent contained up to 1·7 × 106 copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. Significance and Impact of the Study:, The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods. [source] Pitfalls and advantages of different strategies for the absolute quantification of N -acetyl aspartate, creatine and choline in white and grey matter by 1H-MRSNMR IN BIOMEDICINE, Issue 10 2009E. Malucelli Abstract This study extensively investigates different strategies for the absolute quantitation of N -acetyl aspartate, creatine and choline in white and grey matter by 1H-MRS at 1.5,T. The main focus of this study was to reliably estimate metabolite concentrations while reducing the scan time, which remains as one of the main problems in clinical MRS. Absolute quantitation was based on the water-unsuppressed concentration as the internal standard. We compared strategies based on various experimental protocols and post-processing strategies. Data were obtained from 30 control subjects using a PRESS sequence at several TE to estimate the transverse relaxation time, T2, of the metabolites. Quantitation was performed with the algorithm QUEST using two different metabolite signal basis sets: a whole-metabolite basis set (WhoM) and a basis set in which the singlet signals were split from the coupled signals (MSM). The basis sets were simulated in vivo for each TE used. Metabolites' T2s were then determined by fitting the estimated signal amplitudes of the metabolites obtained at different TEs. Then the absolute concentrations (mM) of the metabolites were assessed for each subject using the estimated signal amplitudes and either the mean estimated relaxation times of all subjects (mean protocol, MP) or the T2 estimated from the spectra derived from the same subject (individual protocol, IP). Results showed that MP represents a less time-consuming alternative to IP in the quantitation of brain metabolites by 1H-MRS in both grey and white matter, with a comparable accuracy when performed by MSM. It was also shown that the acquisition time might be further reduced by using a variant of MP, although with reduced accuracy. In this variant, only one water-suppressed and one water-unsuppressed spectra were acquired, drastically reducing the duration of the entire MRS examination. However, statistical analysis highlights the reduced accuracy of MP when performed using WhoM, particularly at longer echo times. Copyright © 2009 John Wiley & Sons, Ltd. [source] Current trends in quantitative proteomicsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2009Monica H. Elliott Abstract It was inevitable that as soon as mass spectrometrists were able to tell biologists which proteins were in their samples, the next question would be how much of these proteins were present. This has turned out to be a much more challenging question. In this review, we describe the multiple ways that mass spectrometry has attempted to address this issue, both for relative quantitation and for absolute quantitation of proteins. There is no single method that will work for every problem or for every sample. What we present here is a variety of techniques, with guidelines that we hope will assist the researcher in selecting the most appropriate technique for the particular biological problem that needs to be addressed. We need to emphasize that this is a very active area of proteomics research,new quantitative methods are continuously being introduced and some ,pitfalls' of older methods are just being discovered. However, even though there is no perfect technique,and a better technique may be developed tomorrow,valuable information on biomarkers and pathways can be obtained using these currently available methods Copyright © 2009 John Wiley & Sons, Ltd. [source] Impaired cytotrophoblast cell,cell fusion is associated with reduced Syncytin and increased apoptosis in patients with placental dysfunctionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2008Manuela Langbein Abstract Preeclampsia (PE), Hemolysis Elevated Liver Enzymes and Low Platelets (HELLP)-syndrome, and intrauterine growth restriction (IUGR) are associated with abnormal placentation. In early pregnancy, placental cytotrophoblasts fuse and form multinuclear syncytiotrophoblasts. The envelope gene of the human endogenous retrovirus-W, Syncytin, is a key factor for mediating cell,cell fusion of cytotrophoblasts. This study investigated clinical parameters of PE and HELLP-associated IUGR and analyzed the cell,cell fusion index and ,-human chorionic gonadotropin (,-hCG) secretion of cytotrophoblasts isolated and cultured from placentas of these patients. In addition, we performed absolute quantitation of Syncytin and determined the apoptosis rate in both cultured cytotrophoblasts and placental tissues. Cultured cytotrophoblasts from PE and HELLP-associated IUGR correlated with a pronounced lower cell,cell fusion index, 1.8- and 3.6-fold; less nuclei per syncytiotrophoblast, 1.4- and 2.0-fold; a significantly decreased ,-hCG secretion, 4.3- and 17.2-fold and a reduction of Syncytin gene expression, 8.1 (P,=,0.019) and 222.7-fold (P,=,0.011) compared with controls, respectively. In contrast, a significantly 2.3-fold higher apoptosis rate was observed in cultured PE/IUGR cytotrophoblasts (P,=,0.043). Importantly, Syncytin gene expression in primary placental tissues of PE/IUGR was 5.4-fold lower (P,=,0.047) and in HELLP/IUGR 10.6-fold lower (P,=,0.019) along with a 1.8- and 1.9-fold significant increase in the apoptosis rate compared with controls, respectively. Low Syncytin expression in both cultured cytotrophoblasts and primary tissues from pathological placentas supports an intrinsic placenta-specific deregulation of cell,cell fusion in the formation of syncytiotrophoblasts leading to increased apoptosis. These processes could contribute to the development and severity of PE and HELLP-associated IUGR. Mol. Reprod. Dev. 75: 175,183, 2008. © 2007 Wiley-Liss, Inc. [source] Pitfalls and advantages of different strategies for the absolute quantification of N -acetyl aspartate, creatine and choline in white and grey matter by 1H-MRSNMR IN BIOMEDICINE, Issue 10 2009E. Malucelli Abstract This study extensively investigates different strategies for the absolute quantitation of N -acetyl aspartate, creatine and choline in white and grey matter by 1H-MRS at 1.5,T. The main focus of this study was to reliably estimate metabolite concentrations while reducing the scan time, which remains as one of the main problems in clinical MRS. Absolute quantitation was based on the water-unsuppressed concentration as the internal standard. We compared strategies based on various experimental protocols and post-processing strategies. Data were obtained from 30 control subjects using a PRESS sequence at several TE to estimate the transverse relaxation time, T2, of the metabolites. Quantitation was performed with the algorithm QUEST using two different metabolite signal basis sets: a whole-metabolite basis set (WhoM) and a basis set in which the singlet signals were split from the coupled signals (MSM). The basis sets were simulated in vivo for each TE used. Metabolites' T2s were then determined by fitting the estimated signal amplitudes of the metabolites obtained at different TEs. Then the absolute concentrations (mM) of the metabolites were assessed for each subject using the estimated signal amplitudes and either the mean estimated relaxation times of all subjects (mean protocol, MP) or the T2 estimated from the spectra derived from the same subject (individual protocol, IP). Results showed that MP represents a less time-consuming alternative to IP in the quantitation of brain metabolites by 1H-MRS in both grey and white matter, with a comparable accuracy when performed by MSM. It was also shown that the acquisition time might be further reduced by using a variant of MP, although with reduced accuracy. In this variant, only one water-suppressed and one water-unsuppressed spectra were acquired, drastically reducing the duration of the entire MRS examination. However, statistical analysis highlights the reduced accuracy of MP when performed using WhoM, particularly at longer echo times. Copyright © 2009 John Wiley & Sons, Ltd. [source] Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome researchPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2007Sebastian Wiese Abstract A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics. [source] Translational and transcriptional analysis of Sulfolobus solfataricus P2 to provide insights into alcohol and ketone utilisationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2007Poh Kuan Chong Abstract The potential of Sulfolobus solfataricus P2 for alcohol or ketone bioconversion was explored in this study. S. solfataricus was grown in different concentrations (0.1,0.8% w/v) of alcohols or ketones (ethanol, iso-propanol, n -propanol, acetone, phenol and hexanol) in the presence of 0.4% w/v glucose. Consequently, the addition of these alcohols or ketones into the growth media had an inhibitory effect on biomass production, whereby lag times increased and specific growth rates decreased when compared to a glucose control. Complete glucose utilisation was observed in all cultures, although slower rates of glucose consumption were observed in experimental cultures (average of 14.9,mg/L/h compared to 18.9,mg/L/h in the control). On the other hand, incomplete solvent utilisation was observed, with the highest solvent consumption being approximately 51% of the initial concentration in acetone cultures. Translational responses of S. solfataricus towards these alcohols or ketones were then investigated using the isobaric tags for relative and absolute quantitation (iTRAQ) technique. The majority (>80%) of proteins identified and quantified showed no discernable changes in regulation compared to the control. These results, along with those obtained from transcriptional analysis of key genes involved within this catabolic process using quantitative RT-PCR and metabolite analysis, demonstrate successful alcohol or ketone conversion in S. solfataricus. [source] Development of an immuno tandem mass spectrometry (iMALDI) assay for EGFR diagnosisPROTEOMICS - CLINICAL APPLICATIONS, Issue 12 2007Jian Jiang Abstract The epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, and is therefore an important biomarker for cancer diagnosis and a target for cancer therapy. We have developed a novel peptide-based immuno tandem mass spectrometry (iMALDI) diagnostic assay for highly sensitive, highly specific, and quantitative analysis of EGFR, which we have applied to the detection of the EGFR peptide in three cell lines and in a tumor biopsy sample. This assay is capable of detecting the EGFR target peptide bound to the antibody beads at attomole levels. The ability to directly obtain amino acid sequence data by MS/MS on any affinity-captured peptides provides specificity to this diagnostic technique. This avoids the problem of "false positives" which can result from the nonspecific binding that can occur with any affinity-based technique. The addition of stable-labeled versions of the target peptide (synthesized from stable-isotope coded amino acids) as internal standards allows absolute quantitation of the target protein. [source] Accelerated tryptic digestion of proteins in plasma for absolute quantitation using a protein internal standard by liquid chromatography/tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2009Fumin Li First page of article [source] | |||||||||||||