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Complex Biological Samples (complex + biological_sample)
Selected AbstractsTowards a specific characterisation of components on a cell surface,combined TERS-investigations of lipids and human cellsJOURNAL OF RAMAN SPECTROSCOPY, Issue 10 2009R. Böhme Abstract Supported lipid structures and human cells (human dermal derived keratinocyte, HaCaT) were investigated using tip-enhanced Raman spectroscopy (TERS) to use the high spatial resolution capabilities of TERS, which is assumed to be less than 10 nm, to determine specific components on the cell surface. As lipids are a main component of cellular membranes, the correlation of spectral properties of pure lipids with respect to the complex biological sample was investigated. Induced by dynamic structural changes as well as nanoscale effects, a particular spectral feature of the lipid TERS spectra is found to vary, and a similar spectral deviation appears among the TERS spectra measured on the cell. Modifications of the cell surface alone cannot cause such behaviour. In contrast to soft lipid agglomerates, the cells were fixed and therefore hampered for intrinsic structural changes. Hence, the main contribution for the cell TERS spectra variation results from nanoscale effects, determined by different spectral characteristics compared to conventional Raman spectroscopy. The present results demonstrate the capability of TERS to provide a detailed and fast insight into the composition of the cell surface, even allowing the detection of single components. Copyright © 2009 John Wiley & Sons, Ltd. [source] Cover Picture: Electrophoresis 3'2010ELECTROPHORESIS, Issue 3 2010Article first published online: 29 JAN 2010 Issue no. 3 is a regular issue with Emphasis on "Proteins and Proteomics". The first part has 8 articles on proteins and proteomics covering various topics, e.g. preparative divergent flow IEF, multichannel gel electrophoresis, capillary gel electrophoresis, nanoparticle-based CE of proteins, 2-DE in a radial gel format, depletion of high abundance proteins, and proteomic investigation of fetal brain and lentil seed. The remaining 10 articles are concerned with nucleic acids, gene expression, methodologies and application. Featured articles include: Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples ((10.1002/elps.200900484)) SDS-PAGE and two-dimensional maps in a radial gel format ((10.1002/elps.200900526)) Analysis of Effect of Electrolyte Types on Electrokinetic Energy Conversion in Nanoscale Capillaries ((10.1002/elps.200900409)) A simple method to determine the surface charge in microfluidic channels ((10.1002/elps.200900603)) [source] Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detectionELECTROPHORESIS, Issue 22 2008Alicia M. Hitchcock Abstract This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1,pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50,mM phosphate buffer, pH 3.5, and reversed polarity at 30,kV with 0.3,psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue. [source] Affinity monolith preconcentrators for polymer microchip capillary electrophoresisELECTROPHORESIS, Issue 16 2008Weichun Yang Abstract Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate- co -ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a >25,000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples. [source] Detection of carbonyl-modified proteins in interfibrillar rat mitochondria using N, -aminooxymethylcarbonylhydrazino- D -biotin as an aldehyde/keto-reactive probe in combination with Western blot analysis and tandem mass spectrometryELECTROPHORESIS, Issue 6 2008Woon-Gye Chung Abstract There is now a large body of supporting data available that links oxidative modifications of proteins to a large number of diseases, degenerative disorders and aging. However, the detailed analysis of oxidative protein modifications remains challenging. Here, we report a new efficient method for identification of oxidatively modified proteins in complex biological samples which is based on the use of an aldehyde-reactive probe, N,-aminooxymethylcarbonylhydrazino- D -biotin (ARP), in combination with Western-type analyses and MS. The biotinylated hydroxylamine derivative forms a chemically stable oxime derivative with the aldehyde/keto group found in carbonyl-modified proteins. The biotin tag is detected by avidin affinity staining. ARP-positive proteins are subsequently subjected to in-gel trypsinization and MS/MS for protein identification. We demonstrate the usefulness of the method for the analysis of protein extracts obtained from interfibrillar heart mitochondria (IFM) from young and old rats. In this study, we identified as putative major protein targets of oxidative modifications the mitochondrial matrix protein, aconitase, the inner mitochondrial membrane protein, ADP/ATP translocase, and constituents of the electron transport chain complexes IV and V. An age-related increase of carbonyl levels was found for aconitase and ATP synthase. [source] Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C,valine isotopic ratios in complex biological samplesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2008Jean-Philippe Godin Abstract On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision 13C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure 13C isotopic enrichment of underivatised amino acids (Asp, Thr,Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(,13C) reported with this method was found to be below 1, . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (,13C = , 12.3 to 150.8,), the calculated root-mean-square (rms) of SD was 0.38 and 0.46, and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (,13C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound 13C,Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p > 0.05). The results of this work indicate that the LC-IRMS was successful for high-precision 13C isotopic measurements in tracer studies giving 13C isotopic enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio. Copyright © 2008 John Wiley & Sons, Ltd. [source] Characterization of drug,protein interactions in blood using high-performance affinity chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 5-6 2009David S. Hage Abstract The binding of drugs with proteins in blood, serum, or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs in the body. High-performance affinity chromatography (HPAC) has received a great deal of interest as a means for studying these interactions. This review examines the various techniques that have been used in HPAC to examine drug,protein binding and discusses the types of information that can be obtained through this approach. A comparison of these techniques with traditional methods for binding studies (e.g., equilibrium dialysis and ultrafiltration) will also be presented. The use of HPAC with specific serum proteins and binding agents will then be discussed, including HSA and ,1 -acid glycoprotein (AGP). Several examples from the literature are provided to illustrate the applications of such research. Recent developments in this field are also described, such as the use of improved immobilization techniques, new data analysis methods, techniques for working directly with complex biological samples, and work with immobilized lipoproteins. The relative advantages and limitations of the methods that are described will be considered and the possible use of these techniques in the high-throughput screening or characterization of drug,protein binding will be discussed. [source] Application of 31P NMR spectroscopy and chemical derivatization for metabolite profiling of lipophilic compounds in human serumMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2009M. Aruni DeSilva Abstract New methods for obtaining metabolic fingerprints of biological samples with improved resolution and sensitivity are highly sought for early disease detection, studies of human health and pathophysiology, and for better understanding systems biology. Considering the complexity of biological samples, interest in biochemical class selection through the use of chemoselective probes for improved resolution and quantitation is increasing. Considering the role of lipids in the pathogenesis of a number of diseases, in this study fingerprinting of lipid metabolites was achieved by 31P labeling using the derivatizing agent 2-chloro-4,4,5,5-tetramethyldioxaphospholane. Lipids containing hydroxyl, aldehyde and carboxyl groups were selectively tagged with 31P and then detected with good resolution using 31P NMR by exploiting the 100% natural abundance and wide chemical shift range of 31P. After standardizing the reaction conditions using representative compounds, the derivatization approach was used to profile lipids in human serum. The results show that the 31P derivatization approach is simple, reproducible and highly quantitative, and has the potential to profile a number of important lipids in complex biological samples. Copyright © 2009 John Wiley & Sons, Ltd. [source] Ultraviolet photofragmentation of biomolecular ionsMASS SPECTROMETRY REVIEWS, Issue 3 2009James P. Reilly Abstract Mass spectrometric identification of all types of molecules relies on the observation and interpretation of ion fragmentation patterns. Peptides, proteins, carbohydrates, and nucleic acids that are often found as components of complex biological samples represent particularly important challenges. The most common strategies for fragmenting biomolecular ions include low- and high-energy collisional activation, post-source decay, and electron capture or transfer dissociation. Each of these methods has its own idiosyncrasies and advantages but encounters problems with some types of samples. Novel fragmentation methods that can offer improvements are always desirable. One approach that has been under study for years but is not yet incorporated into a commercial instrument is ultraviolet photofragmentation. This review discusses experimental results on various biological molecules that have been generated by several research groups using different light wavelengths and mass analyzers. Work involving short-wavelength vacuum ultraviolet light is particularly emphasized. The characteristics of photofragmentation are examined and its advantages summarized. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:425,447, 2009 [source] Click Chemistry-Led Advances in High Content Functional ProteomicsMOLECULAR INFORMATICS, Issue 11-12 2007Abstract The availability of complete genome sequences for numerous eukaryotic and prokaryotic organisms has inspired the advent of new methods to functionally characterize proteins on a global scale. Chemical approaches, in particular, have emerged as a powerful way to investigate the proteome, providing small-molecule probes that report on protein activity and Post-Translational Modification (PTM) state directly in complex biological samples. Many of the key advances made in chemical proteomics can be attributed to the development of efficient bio-orthogonal reactions such as the copper (I)-catalyzed Huisgen's azide,alkyne cycloaddition, a reaction commonly known as "Click Chemistry" (CC). The generation of "clickable" proteomics probes has removed the requirement for bulky reporter tags, thereby allowing access to more biologically relevant systems such as live cells or animals. The versatility of CC has also allowed for greater experimental efficiency, as different reporter tags (i.e., a fluorophore for detection or biotin for enrichment) can be appended to a single probe. Such advances have enabled researchers to identify protein activities dysregulated in disease states, assess the selectivity of enzyme inhibitors in vivo, and inventory specific PTMs on a proteome-wide scale. [source] Selective separation and enrichment of peptides for MS analysis using the microspheres composed of Fe3O4@nSiO2 core and perpendicularly aligned mesoporous SiO2 shellPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2010Hemei Chen Abstract In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe3O4@nSiO2 Core and perpendicularly aligned mesoporous SiO2 shell (designated Fe3O4@nSiO2@mSiO2). The Fe3O4@nSiO2@mSiO2 microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2,nm) and high-surface areas of the microspheres were demonstrated to have good size-exclusion effect for the adsorption of peptides. An increase of S/N ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using myoglobin tryptic digest solution. The enrichment efficiency of re-used Fe3O4@nSiO2@mSiO2 microspheres was also studied. Large-scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano-LC-ESI-MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel Fe3O4@nSiO2@mSiO2 microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples. [source] ICPLQuant , A software for non-isobaric isotopic labeling proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2010Achim Brunner Abstract The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope-coded protein label (ICPL)-labeled peptides on the MS level during LC-MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time-consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS-identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker. [source] Preparation of C60-functionalized magnetic silica microspheres for the enrichment of low-concentration peptides and proteins for MALDI-TOF MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2009Hemei Chen Abstract In this work, for the first time, a novel C60-functionalized magnetic silica microsphere (designated C60-f-MS) was synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres. The resulting C60-f-MS microsphere has magnetite core and thin C60 modified silica shell, which endow them with useful magnetic responsivity and surface affinity toward low-concentration peptides and proteins. As a result of their excellent magnetic property, the synthesized C60-f-MS microspheres can be easily separated from sample solution without ultracentrifuge. The C60-f-MS microspheres were successfully applied to the enrichment of low-concentration peptides in tryptic protein digest and human urine via a MALDI-TOF MS analysis. Moreover, they were demonstrated to have enrichment efficiency for low-concentration proteins. Due to the novel materials maintaining excellent magnetic properties and admirable adsorption, the process of enrichment and desalting is very fast (only 5,min), convenient and efficient. As it has been demonstrated in the study, newly developed fullerene-derivatized magnetic silica materials are superior to those already available in the market. The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel C60-f-MS microspheres makes it a promising candidate for isolation of low-concentration peptides and proteins even in complex biological samples such as serum, plasma, and urine or cell lysate. [source] Facile synthesis of C8 -functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2008Hemei Chen Abstract In this study, novel C8 -functionalized magnetic polymer microspheres were prepared by coating single submicron-sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8 -functionalized magnetic silica (C8 -f-M-S) microspheres exhibit well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8 -f-M-S microspheres provide a convenient and efficient way for enrichment of low-abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI-TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8 -f-M-S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine. [source] A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samplesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2008Sarah Robinson Abstract Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of 18O isotope-based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced 18O from isotopically enriched water into the C-terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof-of-principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples. [source] The use of mass spectrometry for the proteomic analysis of glycosylationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2006Willy Morelle Dr. Abstract Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work. [source] Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometryPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2003Margareta Ramström Abstract The identification and characterization of proteins in complex biological samples such as body fluids, require powerful and reliable tools. Mass spectrometry is today one of the most important methods in such research. This paper reports on the results from the first experiment where a tryptic digest of cerebrospinal fluid was analyzed applying reversed phase liquid chromatography coupled on-line to a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer. In total, 70,204 peaks were detected, which originated from 16,296 isotopic clusters corresponding to 6551 unique peptide masses. From these masses, 39 proteins were identified in the sample. The amount of sample required for one experiment corresponds to 32 ,L of cerebrospinal fluid. [source] Synthesis of S -Adenosyl- L -homocysteine Capture Compounds for Selective Photoinduced Isolation of MethyltransferasesCHEMBIOCHEM, Issue 2 2010Christian Dalhoff Dr. Abstract Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S -adenosyl- L -methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S -adenosyl- L -homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase,cofactor complexes. [source] | ||||||||||