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Digests
Kinds of Digests Selected AbstractsHARM REDUCTION DIGEST 34: How quick to heroin dependence?§DRUG AND ALCOHOL REVIEW, Issue 5 2006ROSS COOMBER In the popular press, and to some extent in the academic literature, there is an assumption that heroin can almost instantly addict a novice user. In this Digest, based on a paper presented at the 2005 APSAD Conference, Coomber & Sutton have extracted quantitative data from their qualitative study of a sample of ,street' heroin users to investigate how rapidly they became physically dependent. They suggest that the period from first use to addiction and regular use to daily use may be longer than many assume and that beliefs about ,instant addiction' are a harm reduction issue. Although small in scope, the study raises questions about the myth of instant heroin addiction which have implications for treatment, prevention and policy. Simon LentonEditor, Harm Reduction Digest [source] HARM REDUCTION DIGEST 12.DRUG AND ALCOHOL REVIEW, Issue 1 2001Harm reduction, drinking patterns, the NHMRC Drinking Guidelines First page of article [source] HARM REDUCTION DIGEST 34: How quick to heroin dependence?§DRUG AND ALCOHOL REVIEW, Issue 5 2006ROSS COOMBER In the popular press, and to some extent in the academic literature, there is an assumption that heroin can almost instantly addict a novice user. In this Digest, based on a paper presented at the 2005 APSAD Conference, Coomber & Sutton have extracted quantitative data from their qualitative study of a sample of ,street' heroin users to investigate how rapidly they became physically dependent. They suggest that the period from first use to addiction and regular use to daily use may be longer than many assume and that beliefs about ,instant addiction' are a harm reduction issue. Although small in scope, the study raises questions about the myth of instant heroin addiction which have implications for treatment, prevention and policy. Simon LentonEditor, Harm Reduction Digest [source] (613) Radiculopathy Treatment Assessment Using Pain Tolerance TestPAIN MEDICINE, Issue 2 2000Article first published online: 25 DEC 200 Authors: Y. Eugene Mironer, Carolinas Center for Advanced Management of Pain; Judson J. Somerville, The Pain Management Clinic of Laredo Current measurements of the outcomes of chronic radiculopathy treatment are limited to subjective criteria: level of pain, range of motion, etc. Our previous study showed that nerve conductivity does not correlate well with the intensity of pain after treatment of radiculopathy (1). In the current study we looked at the Pain Tolerance Threshold (PTT) as a possible measurement of the results of radiculopathy treatment. Twenty patients with chronic radiculopathy (13 lumbar and 7 cervical) underwent epidural steroid injections at the level of involvement. Before, and approximately one week after the procedure, we measured PTT in both the involved and contralateral extremity at 3 different frequencies (5Hz, 250Hz, and 2000 Hz) using Neurometer. Level of pain was also assessed using a Visual Analog Scale (VAS). Initial PTT results showed great interpersonal variability. Nearly half of the patients did not show significant differences in PTT between affected and unaffected sides. Of interest, the majority experienced intolerable pain at 2000 Hz stimulation at lower than maximal intensity output, which contradicts previous findings (2). Dynamics of the PTT measurements after treatment did not directly correlate with changes in the level of pain. Nevertheless, in 7 out of 8 patients with low PTT (relative to the unaffected side) it increased significantly, with noticeable decrease of VAS score. Similar results were not found in patients with either normal initial PTT score or minimal improvement of pain. 1. Mironer YE, Somerville JJ The current perception threshold evaluation in radiculopathy: efficacy in diagnosis and assessment of treatment results. Pain Digest 1998;8:37,38. 2. Liu SS, Gerancher JC, Bainton BG, et al. The effects of electrical stimulation at different frequencies on perception and pain in human volunteers: epidural versus intravenous administration of fentanyl. Anesth Analg 1996;82:98,102. [source] Digest of Articles published in the Annales De Chirurgie 2002, issues 1,10ANZ JOURNAL OF SURGERY, Issue 7 2003Pierre Chapuis DS FRACS First page of article [source] Advanced IMS client supporting secure signalingBELL LABS TECHNICAL JOURNAL, Issue 4 2008Ramana Isukapalli With recent advances in core and access networks and the availability of increased bandwidth and sophisticated devices for end users, there is an increased demand for client applications running on mobile devices, such as laptops and handheld devices, to support real time applications like Voice over Internet Protocol (VoIP) and streaming video, apart from traditional applications like web browsing. This paper presents a prototype IP Multimedia Subsystem (IMS) client, which serves as a VoIP client to set up calls between Internet Protocol (IP) devices and interworks with circuit-switched networks to deliver calls to public switched telephone network (PSTN) phones. It implements supplementary services (including call waiting, call transfer, and call forwarding); supports multimedia ringing, short message service/multimedia messaging service (SMS/MMS), audio/video conferencing, and peer-to-peer video; and it can deliver a call to a user (as opposed to a device) by simultaneously ringing multiple devices registered by the user. Further, to address various security concerns, the client supports Hypertext Transfer Protocol (HTTP) digest authentication using Message Digest 5 (MD5) cryptographic function authentication and key agreement (AKA) and can create secure tunnels to the core network using IP security (IPsec). © 2008 Alcatel-Lucent. [source] The Impact of Underage Drinking Laws on Alcohol-Related Fatal Crashes of Young DriversALCOHOLISM, Issue 7 2009James C. Fell Background:, This study used a pre- to post-design to evaluate the influence on drinking-and-driving fatal crashes of 6 laws directed at youth aged 20 and younger and 4 laws targeting all drivers. Methods:, Data on the laws were drawn from the Alcohol Policy Information System data set (1998 to 2005), the Digests of State Alcohol Highway Safety Related Legislation (1983 to 2006), and the Westlaw database. The Fatality Analysis Reporting System data set (1982 to 2004) was used to assess the ratio of drinking to nondrinking drivers involved in fatal crashes [fatal crash incidence ratio (CIR)]. The data were analyzed using structural equation modeling techniques. Results:, Significant decreases in the underage fatal CIR were associated with presence of 4 of the laws targeting youth (possession, purchase, use and lose, and zero tolerance) and 3 of the laws targeting all drivers (0.08 blood alcohol concentration illegal per se law, secondary or upgrade to a primary seat belt law, and an administrative license revocation law). Beer consumption was associated with a significant increase in the underage fatal CIR. The direct effects of laws targeting drivers of all ages on adult drinking drivers aged 26 and older were similar but of a smaller magnitude compared to the findings for those aged 20 and younger. It is estimated that the 2 core underage drinking laws (purchase and possession) and the zero tolerance law are currently saving an estimated 732 lives per year controlling for other exposure factors. If all states adopted use and lose laws, an additional 165 lives could be saved annually. Conclusions:, These results provide substantial support for the effectiveness of under age 21 drinking laws with 4 of the 6 laws examined having significant associations with reductions in underage drinking-and-driving fatal crashes. These findings point to the importance of key underage drinking and traffic safety laws in efforts to reduce underage drinking-driver crashes. [source] CE coupled to MALDI with novel covalently coated capillariesELECTROPHORESIS, Issue 4 2010Stefan Bachmann Abstract CE offers the advantage of flexibility and method development options. It excels in the area of separation of ions, chiral, polar and biological compounds (especially proteins and peptides). Masking the active sites on the inner surface of a bare fused silica capillary wall is often necessary for CE separations of basic compounds, proteins and peptides. The use of capillary surface coating is one of the approaches to prevent the adsorption phenomena and improve the repeatability of migration times and peak areas of these analytes. In this study, new capillary coatings consisting of (i) derivatized polystyrene nanoparticles and (ii) derivatized fullerenes were investigated for the analysis of peptides and protein digest by CE. The coated capillaries showed excellent run-to-run and batch-to-batch reproducibility (RSD of migration time ,0.5% for run-to-run and ,9.5% for batch-to-batch experiments). Furthermore, the capillaries offer high stability from pH 2.0 to 10.0. The actual potential of the coated capillaries was tested by combining CE with MALDI-MS for analysing complex samples, such as peptides, whereas the overall performance of the CE-MALDI-MS system was investigated by analysing a five-protein digest mixture. Subsequently, the peak list (peptide mass fingerprint) generated from the mass spectra of each fraction was entered into the Swiss-Prot database in order to search for matching tryptic fragments using the MASCOT software. The sequence coverage of analysed proteins was between 36 and 68%. The established technology benefits from the synergism of high separation efficiency and the structure selective identification via MS. [source] On-chip tryptic digest with direct coupling to ESI-MS using magnetic particlesELECTROPHORESIS, Issue 24 2008Anne Le Nel Abstract As a step toward a fully automated front-end microfluidic chip for MS proteomics, we propose a system capable of performing online tryptic digest and ESI-MS, using a replaceable on-chip digestion microcolumn based on self-assembled magnetic particles. [source] Nanostructured copolymer gels for dsDNA separation by CEELECTROPHORESIS, Issue 23 2008Fen Wan Abstract Pluronics are triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) that are able to form many different ordered nanostructures at appropriate polymer concentrations and temperatures in selective solvents. These nanostructured "gels" showed desirable criteria when used as DNA separation media, especially in microchip electrophoresis, including dynamic coating and viscosity switching. A ternary system of F127 (E99P69E99)/TBE buffer/1-butanol was selected as a model system to test the sieving performance of different nanostructures in separating dsDNA by CE. The nanostructures and their lattice constants were determined by small-angle X-ray scattering. Viscosity measurements showed the sol,gel transition phenomena. In addition to the cubic structure, successful electrophoretic separation of dsDNA in 2-D hexagonally packed cylinders was achieved. Results showed that without further optimization, ,X174 DNA,Hae III digest was well separated within 15,min in a 7-cm separation channel, by using F127/TBE/1-butanol gel with a 2-D hexagonal structure. A mechanism for DNA separations by those gels with both hydrophilic and hydrophobic domains is discussed. [source] High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometryELECTROPHORESIS, Issue 21 2003Alexander R. Ivanov Abstract High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 ,m inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n -propanol and formamide as porogens and azobisisobutyronitrile as initiator. N -Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300,000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method. [source] Mass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp.FEBS JOURNAL, Issue 13 2009Nitrogenase Fe-protein modification was analyzed in the endophytic ,-proteobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore, a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide, indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach, we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosylations. [source] A sensitive mutation-specific screening technique for GNAS1 mutations in cases of fibrous dysplasia: the first report of a codon 227 mutation in boneHISTOPATHOLOGY, Issue 6 2007B D Idowu Aims:, To report on the mutation-specific restriction enzyme digest (MSRED) method using paraffin-embedded tissue as a means of detecting GNAS1 mutations in fibrous dysplasia (FD), and to determine if any of the reported GNAS1 mutations in endocrine neoplasms, not previously documented in FD, can be found in FD. Methods and results:, Sixty-seven cases of extragnathic FD were analysed as two groups, 1997,2002 and 2003,06, chosen because tissue fixation and decalcification methods were more accurately recorded in the latter. MSRED revealed that between 2003 and 2006, 93% of 28 ,in house' extragnathic cases harboured a GNAS1 mutation, compared with 75% of 32 cases before 2003. Fixation times of no more than 48 h and decalcification in ethylenediamine tetraacetic acid gave the best results. Of the 56 mutations detected (five gnathic, 51 extragnathic), 32 (57%) were R201H, 21 (38%) were R201C and three (5%) were Q227L. Two Q227L extragnathic cases had unusual clinical/radiological findings. No mutations were detected in osteofibrous dysplasia. Conclusion:, Detection of GNAS1 mutations by MSRED is a valuable adjunct to the histopathological diagnosis of FD. This is the first report of a Q227L mutation in FD, although it has been previously documented in pituitary adenoma. [source] Ethnic variation in AMD-associated complement factor H polymorphism p.Tyr402His,HUMAN MUTATION, Issue 9 2006Michael A. Grassi Abstract Age-related macular degeneration (AMD) is the most common cause of irreversible visual loss in the developed world. Previous studies have demonstrated that the c.1204T>C, p.Tyr402His allelic variant in the complement factor H (CFH) gene is associated with an approximately three-fold increased risk for AMD in Caucasians of predominantly European descent. Both the prevalence as well as the phenotypic spectrum of AMD varies widely among persons of different ethnicities. We hypothesized that populations with a lower prevalence of AMD might also have a lower prevalence of the CFH risk allele. In this study we sought to determine the frequency of this sequence variant in control populations of Caucasians, African Americans, Hispanics, Somalis, and Japanese. Normal control populations were assembled for each ethnic group: Caucasian (n=148), Somali (n=128), African American (n=75), Hispanic (n=81), and Japanese (n=82). Individuals were genotyped using a restriction digest assay and the frequency of the C allele at nucleotide position 1204 of the CFH gene was determined. A bioinformatic approach was used to identify SNPs in linkage disequilibrium with rs1061170 (c.1204T>C, p.Tyr402His) from the human haplotype map project database (HapMap) in order to validate the findings. We found widely discordant frequencies of the risk allele between some of the different ethnic groups: Japanese 0.07±0.02, Hispanics 0.17±0.03, African-Americans 0.35±0.04, Caucasians 0.34±0.03, and Somalis 0.34±0.03. Allele frequencies generated by analysis of the HapMap database were consistent with these findings. This study suggests that there are other yet unidentified genetic factors important in the pathogenesis of AMD that may mitigate the effects of c.1204T>C, p.Tyr402His variant. Hum Mutat 27(9), 921,925, 2006. © 2006 Wiley-Liss, Inc. [source] Fruit and fibre: the nutritional value of figs for a small tropical ruminant, the blue duiker (Cephalophus monticola)AFRICAN JOURNAL OF ECOLOGY, Issue 4 2009Erin L. Kendrick Abstract Tropical forests throughout the world are home to a guild of small ruminants that consume fruit as a substantial portion of their diet. Because the rumen is relatively inefficient at digesting nonstructural carbohydrates and only slowly digests cellulose, the feeding adaptations of frugivorous ruminants are enigmatic. We examined the nutritional value of wild figs to blue duikers, one of the smallest and most frugivorous ruminants, through chemical analyses and a series of digestion trials with six species of wild African figs. These figs were high in fat, protein, cell wall, lignin and Ca : P ratios, low in sugar and starch, and high in unextractable, fibre-bound tannins when compared with many other fruits. The fibre-bound tannins and protein caused protein digestibility and nitrogen balance to be consistently low or negative. The high fibre content of the figs allowed duikers to only digest 30,60% of energy contained in the figs. However, duikers were able to consume enough digestible energy to maintain body mass during 4-day trials. Therefore, a ruminant digestive system is beneficial to mammals eating high fibre, high-tannin tropical fruit like figs, especially if the mammal is small enough to harvest a sufficient amount to meet its daily energy requirements and has adaptations for reducing the effects of tannins on protein digestibility. Résumé Les forêts tropicales du monde entier abritent toute une faune de petits ruminants dont les fruits constituent une part non négligeable de l'alimentation. Comme le rumen est relativement peu efficace pour digérer les hydrates de carbone non structuraux et ne digère que lentement la cellulose, les adaptations alimentaires des ruminants frugivores sont encore énigmatiques. Nous avons examiné la valeur nutritionnelle des figues sauvages pour le céphalophe bleu, un des ruminants les plus petits et parmi les plus frugivores, au moyen d'analyses chimiques et d'une série d'essais de digestion avec six espèces de figues sauvages africaines. Ces figues étaient riches en graisses, en protéines, en parois cellulaires, en lignine, et leur rapport Ca/P était élevé; elles avaient un contenu faible en sucre et en amidon, et beaucoup de tanins impossibles à extraire, liés aux fibres, comparés à de nombreux autres fruits. Les tanins liés aux fibres et les protéines faisaient que la digestibilité des protéines et l'équilibre azotéétaient en permanence faibles ou négatifs. Le contenu en fibres élevé des figues ne permettait aux céphalophes de digérer que 30 à 60% de l'énergie contenue dans les figues. Cependant, pendant les quatre jours du test, les céphalophes ont pu consommer suffisamment d'énergie digestible pour conserver leur masse corporelle. C'est pourquoi le système digestif des ruminants est bénéfique pour les mammifères qui consomment des fruits tropicaux riches en fibres et en tanins, comme les figues, spécialement si le mammifère est assez petit pour pouvoir en trouver une quantité suffisante pour répondre à ses besoins quotidiens en énergie, et qu'il possède des adaptations qui lui permettent de réduire les effets des tanins sur la digestibilité des protéines. [source] Classification and Antihypertensive Activity of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Food ProteinsJOURNAL OF FOOD SCIENCE, Issue 4 2000H Iroyukifujita ABSTRACT: Angiotensin I-converting enzyme (ACE)-inhibitory peptides from the thermolysin digest of chicken muscle and the peptic digest of ovalbumin were isolated. However, some of them failed to show antihypertensive activity in spontaneously hypertensive rats (SHR). To clarify this discrepancy, ACE-inhibitory peptides from various sources were preincubated with ACE before measurement of ACE-inhibitory activity and classified into 3 groups: (1) inhibitor type, IC50 values of peptides that are not affected after preincubation with ACE; (2) substrate type, peptides that are hydrolyzed by ACE to give peptides with weaker activity; and (3) prodrug-type inhibitor, these peptides are converted to true inhibitors by ACE or gastrointestinal proteases. Peptides belonging to the 1st and the 3rd groups exert antihypertensive activities even after oral administration in SHR. [source] HPTLC/DESI-MS imaging of tryptic protein digests separated in two dimensions,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2008Sofie P. Pasilis Abstract Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the corresponding proteins were identified using a protein database search software. Two-dimensional distributions of identified peptides were mapped for each separated protein digest. Sequence coverages for cytochrome c and myoglobin were 81 and 74%, respectively. These compared well with those determined using the more standard HPLC/ESI-MS/MS approach (89 and 84%, respectively). Preliminary results show that use of more sensitive instrumentation has the potential for improved detection of peptides with low Rf values and improvement in sequence coverage. However, less multiple charging and more sodiation were seen in HPTLC/DESI-MS spectra relative to HPLC/ESI-MS spectra, which can affect peptide identification by MS/MS. Methods to increase multiple charging and reduce the extent of sodiation are currently under investigation. Published in 2008 by John Wiley & Sons, Ltd. [source] Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2003Toshiyuki Kosaka Abstract We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-,-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging. Copyright © 2003 John Wiley & Sons, Ltd. [source] Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibrationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2002Omar Belgacem Abstract Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de- N -glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of ±0.12 and ±0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip® technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III, and AT-III,, were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de- N -glycosylation (by means of PNGase F) of the ,- and ,-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (,0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N - and O -glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous ,-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N -glycans. Copyright © 2002 John Wiley & Sons, Ltd. [source] Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2008Lucie Korecká Abstract We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG),Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach. [source] Assessing a novel microfluidic interface for shotgun proteome analysesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007An Staes Abstract Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage. [source] Design and synthesis of a solid-phase fluorescent mass tagJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2005Yang Shi Abstract In this study, we demonstrate the design of a new solid-phase fluorescent mass tag (FMT) that contains the following features: (1) the FMT is synthesized using Fmoc chemistry which is simple, rapid, and cost-effective; (2) lysine is used as a uniformly labeled amino acid (using stable isotopes) to allow 8 Da difference between "heavy" and "light" tags; (3) a fluorescent molecule is coupled to the isotope tag that allows a tagged peptide to be detected by online fluorescence; and (4) an iodoacetyl reactive group provides cysteine reactivity. Using MALDI-TOF MS and HPLC, we show that the FMT reagent can be used to label standard cysteine-containing peptides as well as cysteine-containing peptides from a BSA tryptic digest. [source] Nutrient content and yield in relation to top breakover in onion developed from greenhouse-grown transplants,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 5 2009Vincent M Russo Abstract BACKGROUND: Onions (Allium cepa L.) generally are harvested based on percentage of tops broken over. Since plant metabolism changes over time, percentage of tops broken over may be used to determine a harvest time to deliver marketable bulbs with the best nutrient content. RESULTS: The cultivars Candy and Texas Grano 1015 Y were harvested at 10%, 20%, 30%, 40% and 50% breakover in 2006 and 2007. Larger and heavier bulbs were produced by Candy and in 2006, the year with near-normal precipitation. There was little difference in bulb size and weight due to percent breakover. Contents of chemical moieties in bulbs were affected by year, with the majority of values being higher in 2006, and there were either no differences due to cultivar, or where differences were found nitrate-N, phosphate and sulfate contents were lower in Candy. Soluble solids content was lower in 2006 and higher in Candy. Content of nitrogen and phosphorous in a Kjeldahl digest, nitrate-N, phosphate, potassium and sulfate were either linearly or quadratically distributed over percent breakover. Nitrite-N, calcium, magnesium, sodium and soluble solids were randomly distributed over percent breakover. Bulb size and weight did not change from the 20% breakover point, and most of the chemical moieties analyzed, with the exception of nitrate- and nitrite-N values, were highest below the 30% breakover. CONCLUSION: Harvest occurring soon after breakover begins could be beneficial in terms of nutrient content without loss of bulb size or weight. Copyright © 2009 Society of Chemical Industry [source] Simulated digest of a glucosamine-based equine nutraceutical modifies effect of IL-1 in a cartilage explant model of inflammationJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2008W. PEARSON First page of article [source] Fate of 14C-acrylamide in roasted and ground coffee during storageMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 5 2008Matthias Baum Abstract Acrylamide (AA) is formed during heating of carbohydrate rich foods in the course of the Maillard reaction. AA has been classified as probably carcinogenic to humans. Storage experiments with roasted coffee have shown that AA levels decrease depending on storage time and temperature. In the present study the fate of AA lost during storage of roasted and ground (R&G) coffee was studied, using 14C-labeled AA as radiotracer. Radiolabel was measured in coffee brew, filter residue, and volatiles. In the brew, total 14C-label decreased during storage of R&G coffee, while activity in the filter residue built up concomitantly. [2,3- 14C]-AA (14C-AA) was the only 14C-related water extractable low molecular compound in the brew detected by radio-HPLC. No formation of volatile 14C-AA-related compounds was detected during storage and coffee brewing. Close to 90% of the radiolabel in the filter residue (spent R&G coffee, spent grounds) remained firmly bound to the matrix, largely resisting extraction by aqueous ammonia, ethyl acetate, chloroform, hexane, and sequential polyenzymatic digest. Furanthiols, which are abundant as aroma components in roasted coffee, have not been found to be involved in the formation of covalent AA adducts and thus do not contribute substantially to the decrease of AA during storage. [source] Rapid selection of peptide containing fractions in off-line 2-D HPLC in shotgun proteome analysis by screening with MALDI TOF MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2009Maria Lasaosa A simple and fast screening method for the selection of fractions of first dimension separation to be analyzed in second dimension-MS/MS experiments in offline multidimensional liquid chromatographic separation schemes for shotgun proteome analysis was developed. The method is based on the measurement of total peptide content of the first dimension fractions by MALDI MS and was established using a tryptic digest of a bacterial proteome. The results of the screening process were in good agreement with those obtained in a detailed proteome analysis performed by RP×ion-pair RP-MALDI TOF/TOF MS analysis. The method supports a straightforward planning of experiments, also enabling a reduction of overall measurement time in shotgun proteome analysis. [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] Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatographyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2008Guanghui Han Abstract The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe3+ immobilized metal affinity chromatography (Fe3+ -IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe3+ -IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%. [source] Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap columnPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2007Xiaogang Jiang Abstract An approach was developed to automate sample introduction for nanoflow LC-MS/MS (,LC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100,,m id×2,cm SCX trap column and a 75,,m id×12,cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current ,LC-MS/MS system. This system represented a promising platform for routine proteome analysis. [source] | ||||||||||||||||||||||||||||||||||