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Sample Preparation Methods (sample + preparation_methods)
Selected AbstractsEFFECTS OF DIFFERENT SAMPLE PREPARATION METHODS ON STABLE CARBON AND OXYGEN ISOTOPE VALUES OF BONE APATITE: A COMPARISON OF TWO TREATMENT PROTOCOLS*ARCHAEOMETRY, Issue 1 2010C. J. YODER Researchers have long debated the appropriateness of stable isotope analysis of bone apatite to reconstruct the diets of ancient animals. The debate has centred, in part, on diagenesis of bone mineral from interaction with the burial environment. A number of acetic acid treatments are used to remove diagenetic carbonates from samples; however, less is known on how different protocols alter stable isotope values. We compare two common acetic acid solution treatments (0.1 M versus 1.0 M-buffered) to examine the effects on carbon and oxygen isotope values and Fourier transform infrared spectroscopy (FTIR) spectra in human bone from different burial contexts. Results indicate that both treatments have a similar effect on isotope values and FTIR spectra in bone apatite. [source] Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activitiesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2001Min-Jung Kang Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r2,=,0.9998) over two orders of magnitude of concentration of rac -1-phenylethylamine (PEA), which is converted to 2-methoxy- N -[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (,5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes. Copyright © 2001 John Wiley & Sons, Ltd. [source] Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urineELECTROPHORESIS, Issue 14 2005Dan Theodorescu Dr. Abstract We describe the use of capillary eletrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47,patients who underwent prostate biopsy. Of this group, 21,patients had benign pathology and 26 with,CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47,urine samples were compared to that of 41,young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474,samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP. [source] A comparison of EDI with solvent-free MALDI and LDI for the analysis of organic pigmentsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2008Ichiro Kudaka Abstract To evaluate the applicability of EDI to material analysis as a new ionization method, a comparison of EDI with solvent-free matrix-assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) was made for the analysis of organic pigments, e.g. Pigment Yellow 93, Pigment Yellow 180, and Pigment Green 36, as test samples, which are poorly soluble in standard solvents. In EDI, the samples were prepared in two ways: deposition of suspended samples in appropriate solvents and dried on the substrate, and the direct deposition of the powder samples on the substrate. No matrices were used. Both sample preparation methods gave similar mass spectra. Equally strong signals of [M + H]+ and [M , H], ions were observed with some fragment ions for azo pigments in the respective positive or negative mode of operation. For the powder sample of the phthalocyanine pigment PG36, M+, and [M + H]+ in the positive mode and M,, in the negative mode of operation were observed as major ions. Positive-mode, solvent-free MALDI gave M+, [M + H]+ and [M + Na]+ and negative mode gave [M , H], depending on the sample preparation. As solvent-free MALDI, EDI was also found to be an easy-to-operate, versatile method for the samples as received. Copyright © 2008 John Wiley & Sons, Ltd. [source] Characterization of N -palmitoylated human growth hormone by in situ liquid,liquid extraction and MALDI tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2007Emmanuelle Sachon Abstract Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N, group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid,liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N -palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N -palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications. Copyright © 2007 John Wiley & Sons, Ltd. [source] The minotaur proteome: Avoiding cross-species identifications deriving from bovine serum in cell culture modelsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2010Jakob Bunkenborg Abstract Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5,15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented. [source] Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2009Anne K. Callesen Abstract Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous sample collection and sample handling protocols. The sensitivity of the MS analysis relies on the quality of the sample; consequently, the blood sample preparation step is crucial to obtain pure and concentrated samples and enrichment of the proteins and peptides of interest. This review focuses on the serum sample preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE sample preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting. [source] Maximized PUFA measurements improve insight in changes in fatty acid composition in response to temperatureARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Coby van Dooremalen Abstract A general mechanism underlying the response of ectotherms to environmental changes often involves changes in fatty acid composition. Theory predicts that a decrease in temperature causes an increase in unsaturation of fatty acids, with an important role for long-chain poly-unsaturated fatty acids (PUFAs). However, PUFAs are particularly unstable and susceptible to peroxidation, hence subtle differences in fatty acid composition can be challenging to detect. We determined the fatty acid composition in springtail (Collembola) in response to two temperatures (5°C and 25°C). First, we tested different sample preparation methods to maximize PUFAs. Treatments consisted of different solvents for primary lipid extraction, mixing with antioxidant, flushing with inert gas, and using different temperature exposures during saponification. Especially slow saponification at low temperature (90,min at 70°C) in combination with replacement of headspace air with nitrogen during saponification and methylation maximized PUFAs for GC analysis. Applying these methods to measure thermal responses in fatty acid composition, the data showed that the (maximized) proportion of C20 PUFAs increased at low acclimation temperature. However, C18 PUFAs increased at high acclimation temperature, which is contrary to expectations. Our study illustrates that PUFA levels in lipids may often be underestimated and this may hamper a correct interpretation of differential responses of fatty acid composition. © 2009 Wiley Periodicals, Inc. [source] | ||||||||||