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Amino Acid Analysis (amino + acid_analysis)
Selected AbstractsPurification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airagJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2006B. Batdorj Abstract Aims:, The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. Methods and Results:, Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2,10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and , -chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N -termini were blocked hampering straightforward Edman degradation. Conclusions:, Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. Significance and Impact of the Study:, Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998). [source] Nitrate modifies the assimilation pattern of ammonium and urea in wheat seedlingsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 3 2010Maria Garnica Abstract BACKGROUND: In certain plant species, ammonium or urea nutrition can cause negative effects on plant development which can result in toxic symptoms. Some authors suggest that the presence of nitrate can alleviate these symptoms by increasing ammonium and urea assimilation, avoiding its accumulation. In order to study this hypothesis, wheat (Triticum aestivum L.) seedlings were grown with various nitrogen supplies containing the main nitrogen forms (ammonium, nitrate and urea). Amino acids content and the activity of the three main enzymes involved in nitrogen assimilation (nitrate reductase, glutamine synthetase and urease) were studied. RESULTS: The application of nitrate along with urea and/or ammonium was not associated with a time-sustained increase in the activity of glutamine synthetase and urease. Amino acid analysis revealed that nitrate induced changes in amino acid metabolism enhancing its concentration. Likewise the content of protein was also higher in nitrate-treated plants. CONCLUSION: These results suggest that the effect of nitrate is compatible with a rapid and transient increase in the activity of glutamine synthetase and urease during the first hour after the onset of treatments. Nevertheless, a possible effect of nitrate reducing ammonium accumulation through the activation of alternative metabolic pathways different from that involving glutamine synthetase cannot be ruled out. Finally, nitrate effects on amino acid concentration indicate that, whereas ammonium assimilation takes place principally in the root, urea and nitrate assimilation occurred in the shoot, under the conditions of the experiment. Copyright © 2009 Society of Chemical Industry [source] Replacement of Fish Meal with Poultry By-product Meal as a Protein Source in Pond-raised Sunshine Bass, Morone chrysops , × M. saxatlis ,, DietsJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 5 2008Harvey J. Pine Replacement of fish meal (FM) as a protein source with alternative sources of protein in aquaculture diets has been widely explored in aquaculture. The goal of replacement of FM in production diets is to maintain growth, lower production costs, and increase sustainability. Evaluation of the replacement of FM with poultry by-product meal (PBM) in phase II sunshine bass diets, Morone chrysops × M. saxatilis, was conducted in ponds over 246 d. Four diets were formulated to be isonitrogenous (37%) and isocaloric (4 kcal/g) with different levels of FM replacement with PBM (0, 33, 67, and 100%, Diets 1,4, respectively). Twelve ponds were stocked with 400 phase II sunshine bass (mean weight 5.6 g) and randomly assigned one of the four diets. Fish were fed below satiation based on predicted growth and feed conversion, initially once daily (1700 h) and then twice daily (0700 and 1700 h) as water temperatures and feeding activity increased. Diets were evaluated based on production and performance indicators, body composition, and economic analysis. Production results revealed no significant differences in mean final individual fish weight (511 ± 21 g), net production (4257 ± 247 kg/ha), and survival (85 ± 2%). No significant differences occurred between the performance indicators: mean feed conversion ratio (2.47 ± 0.11), specific growth rate (1.84 ± 0.02), and protein conversion efficiency (23 ± 1.3%). Body composition was statistically similar for mean percent fillet weight (49 ± 0.6%) and percent intraperitoneal fat (9.8 ± 1.0%); however, the hepatosomatic index was significantly different between Diets 3 (3.7 ± 0.1%) and 4 (3.2 ± 0.1%). Mean proximate analysis of whole fish (dry weight basis) was not significantly different among treatments yielding the following: percent protein (46 ± 0.4%), lipid (47 ± 1.3%), and ash (8 ± 0.7%). Mean fillet composition (dry weight basis) also revealed no significant differences: percent protein (72 ± 0.8%), percent lipid (30 ± 1.6%), and percent ash (5 ± 0.2%). Proximate analysis was also performed on the diets and revealed a significantly lower protein content in Diet 3 (34.3 ± 0.5%) compared to the other diets (37.1 ± 0.4%). Amino acid analysis of the diets indicated a possible deficiency in methionine in Diets 3 and 4. Based on production, performance, and body composition, the results indicate that complete replacement of FM with PBM in sunshine bass diets is feasible; however, economic analysis suggests that the replacement of FM with PBM may result in reduced revenue over feed costs. [source] Trichothiodystrophy-like Hair Abnormalities in a Child with Keratitis Ichthyosis Deafness SyndromePEDIATRIC DERMATOLOGY, Issue 4 2008L. De Raeve M.D., Ph.D. It appears to be genetically heterogeneous and may be caused by mutations in the connexin 26 (Cx26) gene (GJB2) or in the connexin 30 gene. It is characterized by the association of ichthyosis-like skin lesions, hearing loss, and vascularizing keratitis. We report the clinical and molecular findings in a 5-year-old girl with keratitis ichthyosis deafness syndrome. DNA sequencing in our patient revealed a p.Ser17Phe mutation in GJB2. Besides the typical clinical features of keratitis ichthyosis deafness syndrome, a peculiar intriguing finding not previously described in the literature in this condition was that polarizing light microscopy of the scalp hair in our patient revealed striking bright and dark bands as seen in trichothiodystrophy. Amino acid analysis of the hair sample also disclosed a reduced cysteine index. We emphasize that it would be of great benefit to examine hair shafts in other patients with keratitis ichthyosis deafness syndrome for trichothiodystrophy-like abnormalities. [source] Recent advances in amino acid analysis by CEELECTROPHORESIS, Issue 1 2008Véréna Poinsot Abstract This paper describes a number of articles that have been published on amino acid (AA) analysis using CE during the period from June 2005 to May 2007. This review article follows the format of the previous articles of Smith (Electrophoresis 1999, 20, 3078,3083), Prata et al.. (Electrophoresis 2001, 22, 4129,4138), and Poinsot et al.. (Electrophoresis 2003, 24, 4047,4062 and Electrophoresis 2006, 27, 176,194). Several new developments in AA analysis with CE are reported describing the use of laser-emitting diodes for LIF, MS, and chips. In addition, we describe articles concerning clinical studies and neuroclinical applications. [source] Determination of amino acids by micellar EKC: Recent advances in method development and novel applications to different matricesELECTROPHORESIS, Issue 1 2008Paolo Iadarola Professor Abstract The extensive use of CE for the analysis of amino acids has been well documented in a series of research articles and reviews. Aim of this report is to address the attention of the reader on the recent advances of micellar electrokinetic chromatography for the separation and determination of these analytes. Enhancements in selectivity of this technique through the use of pseudostationary phases containing mixed micelles, polymers, and chiral selectors are presented. Selected applications concerning separation and quantitation of even minute amounts of amino acids in: (i) biological fluids; (ii) microdialysates; (iii) plant cells; (iv) food stuff; and (v) pharmaceutical formulations have also been covered. Advantages of MEKC over other techniques for the amino acid analysis have been underlined. [source] Recent advances in amino acid analysis by capillary electrophoresisELECTROPHORESIS, Issue 22-23 2003Véréna Poinsot Abstract Amino acids are studied extensively using capillary electrophoresis. In a previous article, we reviewed applications reported in the period 1999 , early 2001 (Prata, C., Bonnafous, P., Fraysse, N., Treilhou, M., Poinsot, V., Couderc, F., Electrophoresis 2001, 22, 4129,4138). In this article we follow on with this review for the period end of 2001 , beginning of 2003. We will report the developments of detection methods, separations of enantiomers, the new medical applications, and amino acids in food and plants. This review shows that CE is more and more important for the amino acid analysis. [source] Valproic Acid-Induced Hyperammonemic Encephalopathy with Triphasic WavesEPILEPSIA, Issue 7 2000Akira Kifune Summary: Purpose: To examine a patient with valproic acid (VPA)-induced hyperammonemic encephalopathy accompanied by triphasic waves. Methods: A 61-year-old male patient with epilepsy experienced disturbance of consciousness after VPA dose was increased because of poor seizure control. The electroencephalogram (EEG) taken on admission revealed triphasic waves and high-amplitude ,-activity with frontal predominance. Although serum hepatic enzymes, such as AST and ALT, were normal, serum ammonium level was high at 96 ,g/dl (normal range, 3,47 ,g/dl). Serum amino acid analysis showed multiple minor abnormalities. Administration of VPA was discontinued immediately after admission, while other anticonvulsants were continued. Results: The patient's condition was improved on the fourth day of admission. An EEG, serum ammonium level, and amino acid profile were normal on the eighth day. Based on VPA administration, serum ammonium levels, and results of amino acid analysis, this patient had VPA-induced hyperammonemic encephalopathy. Conclusions: Our case indicates that caution is required if triphasic waves appear in VPA-induced hyperammonemic encephalopathy. [source] How do enamelysin and kallikrein 4 process the 32-kDa enamelin?EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2006Yasuo Yamakoshi The activities of two proteases , enamelysin (MMP-20) and kallikrein 4 (KLK4) , are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion. [source] Assignment of a single disulfide bridge in rat liver methionine adenosyltransferaseFEBS JOURNAL, Issue 1 2000María L. Martínez-Chantar Rat liver methionine adenosyltransferase incorporated 8 mol of N -ethylmaleimide per mol of subunit upon denaturation in the presence of 8 m urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme. [source] Flame retardancy finish with an organophosphorus retardant on silk fabricsFIRE AND MATERIALS, Issue 6 2006Jin-Ping Guan Abstract The paper mainly deals with flame retardancy of silk fabrics treated with a commercial organophosphorus flame retardant [N-hydroxymethyl (3-dimethyl phosphono) propionamide (HDPP), also known as Pyrovatex CP], using the pad-dry-cure-wash method. The structures and properties of the treated and control sample are discussed. The Limiting Oxygen Index (LOI) value of the modified sample is above 30%. After 50 laundry cycles, it still has some flame retardancy left. HDPP and a cross-linking agent (HMM) were bound to silk fabrics which is confirmed by FT-IR spectra and amino analysis. The reaction degree of the flame retardant with silk is also high; almost all the tyrosine units have reacted, which can be confirmed by amino acid analysis. The reaction between flame retardant and silk only occurs in the amorphous region of silk fibre, which is confirmed by X-ray diffraction analysis and amino acid analysis. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis show that the flame retardant causes silk fabrics to decompose below its ignition temperature (600°C) and formed carbonaceous residue or char when exposed to fire. The char behaves as a thermal barrier to fire, so silk fabrics show good flame retardancy. The treatment has a little effect on the whiteness of the silk fabrics and the tensile strength of treated silk fabrics slightly decreased; both effects are negligible. Copyright © 2006 John Wiley & Sons, Ltd. [source] Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus speciesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2001Gerold Bacher Abstract A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn. muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS). After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography. Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis. Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix ,-cyano-4-hydroxycinnamic acid. The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8,10 pmol range and with a mass accuracy of ±0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice. After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data. Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity. Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides. Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information. Three examples are presented: (a) CID MSn (n = 2,4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B. subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2,4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B. subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B. megaterium. All MSn experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages. MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions. In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra. The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms). The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique. Copyright © 2001 John Wiley & Sons, Ltd. [source] The oxidation metabolites of endomorphin 1 and its fragments induced by free radicalsJOURNAL OF PEPTIDE SCIENCE, Issue 5 2009Pin Gong Abstract Endomorphin 1 (EM1), an endogenous µ-opioid receptor agonist, acts as a free radical scavenger in vitro and an antioxidant in vivo. The modification of EM1 by ROS and the properties of the OM attracted our attention. In vitro assays were performed via RP-HPLC, spectrophotometric measurements, EPR and amino acid analysis, Schmorl's reaction to define the formation of melanin-like compounds transformed from EM1, collectively named EM1,melanin and by solubility assay, radioligand-binding assay, NADH oxidation, superoxide anion scavenging assay to study some physical and chemical properties of EM1,melanin. Possible pathways of the formation of EM1,melanin were proposed. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Solid phase peptide synthesis on epoxy-bearing methacrylate monolithsJOURNAL OF PEPTIDE SCIENCE, Issue 12 2004E. Vlakh Abstract Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing ,-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] Synthesis, conformation and biological activity of linear and cyclic Thr6 -bradykinin analogues containing N -benzylglycine in place of phenylalanine,JOURNAL OF PEPTIDE SCIENCE, Issue 12 2001L. Biondi Abstract Three linear Thr6 -bradykinin analogues in which either one or both the two phenylalanine residues in the peptide sequence have been substituted by N -benzylglycine (BzlGly) and their head-to-tail cyclic analogues were synthesized and tested on an isolated rat duodenum preparation. The linear (BzlGly5,Thr6 -BK, BzlGly8,Thr6 -BK and BzlGly5,8,Thr6 -BK) and the cyclic (cyclo BzlGly5,Thr6 -BK, cyclo BzlGly8,Thr6 -BK and cyclo BzlGly5,8,Thr6 -BK) peptoid-like analogues were characterized by amino acid analysis, optical rotation, analytical HPLC and MALDI-TOF mass spectroscopy. The conformational features of both the linear and cyclic derivatives were investigated by FT-IR and CD measurements. Preliminary molecular mechanics calculations were also performed on some synthetic peptides. Pharmacological screening using the relaxation of the isolated rat duodenum preparation showed that incorporation of N -benzylglycine at positions 5 and/or 8 in the linear Thr6 -BK causes a substantial decrease in potency. Comparable incorporation in cyclo Thr6 -BK, at position 8, or 5 and 8, resulted in nearly inactive analogues. However, cyclo BzlGly5,Thr6 -BK showed a potency which is of the same order of magnitude as for cyclo -BK and cyclo Thr6 -BK. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Properties and Bioapplications of Blended Cellulose and Corn Protein FilmsMACROMOLECULAR BIOSCIENCE, Issue 9 2009Quanling Yang Abstract A series of blend films have been prepared from cellulose and corn protein in a NaOH/urea solution by a simple, low cost, and ,green' pathway. Their structure and properties are characterized by amino acid analysis, X-ray diffraction, scanning electron microscopy, thermogravimetry, and tensile testing. The results reveal that a certain miscibility exists between cellulose and corn protein and their thermal stability and mechanical properties are improved significantly, compared with the protein materials, when the protein content is less than 18 wt.-%. The protein, which contains tyrosine and histidine, could remain in the blend films after being washed for ten days, which indicates the strong hydrogen bonding between the hydroxy groups of cellulose and the hydroxyphenyl of tyrosine and imidazolyl of histidine in the protein. Furthermore, they exhibit good biocompatibility capable of supporting cell adhesion and proliferation. [source] Standardization of allergen products: 1.ALLERGY, Issue 7 2009Detailed characterization of GMP-produced recombinant Bet v 1.0101 as biological reference preparation Background:, Standardization of allergen extracts requires the availability of well-characterized recombinant allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. Methods:, Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. Results:, Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4°C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. Conclusion:, Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1. [source] Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detectionRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2008Dennis J. Dietzen Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20,min. The dynamic range of each amino acid was generally 1,1000,µmol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88,125%. Deming regression with the Beckman 7300 yielded slopes from 0.4,1.2, intercepts from ,21 to 65,µmol/L, correlation coefficients from 0.84,0.99 and Syx from 2,125,µmol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, , -alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors. Copyright © 2008 John Wiley & Sons, Ltd. [source] Characterisation of combinatorial libraries of mucin-2 antigen peptides by high-resolution mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2002Emöke Windberg An epitope motif, TX1TX2T, of mucin-2 glycoprotein was identified by means of a mucin-2-specific monoclonal antibody, mAb 994, raised against a synthetic mucin-derived 15-mer peptide conjugate. For determination of the epitope sequence recognised with highest affinity by mAb 994, a combinatorial approach was applied using the portioning-mixing technique excluding Cys. Antibody binding of libraries was most profound when Gln was at the X1 position. Analytical characterisation of the TQTX2T library was conducted by amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and electrospray ionisation Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometric methods. Control libraries were prepared by mixing 19 individual peptides corresponding to the TQTX2T sequence. Thus, mixtures of 6, 10 and 19 pentapeptides were analysed and compared with the combinatorial mixture. MALDI-TOFMS was able to detect only partially the components in the 6- and 10-member mixtures, but failed to characterise a more complex 19-member mixture. In contrast, ESI-FTICRMS resolved all mixtures of higher complexity and provided direct identification at monoisotopic resolution, such as for a peptide library containing ,isobaric' lysine and glutamine (,m,=,0.0364,Da). The results of this study suggest that ESI-FTICRMS is a powerful tool for characterisation of combinatorial peptide libraries of higher complexity. Copyright © 2002 John Wiley & Sons, Ltd. [source] Structure, biological activity and membrane partitioning of analogs of the isoprenylated a -factor mating peptide of Saccharomyces cerevisiaeCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2000H. Xie Abstract: Previous biochemical investigations on the Saccharomyces cerevisiaea -factor indicated that this lipopeptide pheromone [YIIKGVFWDPAC(farnesyl)OMe] might adopt a type II ,-turn at positions 4 and 5 of the peptide sequence. To test this hypothesis, we synthesized five analogs of a -factor, in which residues at positions 4 and 5 were replaced with: l -Pro4(I); d -Pro4(II); l -Pro4 - d -Ala5(III); d - Pro4 - l -Ala5(IV); or Nle4(V). Analogs were purified to > 99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis. Using a growth arrest assay the conformationally restricted a -factor analogs I and III were found to be almost 50-fold more active than the diastereometric homologs II and IV and were equally active to wild-type a -factor. Replacement of Lys4 with the isosteric Nle4 almost abolished the activity of the pheromone. Thus, the incorporation of residues that promote a type II ,-turn compensated for the loss of the favorable contribution of the Lys4 side chain to pheromone activity. CD spectra on these peptides suggested that they were essentially disordered in both TFE/H2O and in the presence of DMPC vesicles. There was no correlation between CD peak shape and biological activity. Using fluorescence spectroscopy we measured the interaction of lipid vesicles with these position 4 and 5 analogs as well as with three a -factor analogs with a modified farnesyl group. The results indicated that modifications of both the peptide sequence and the lipid moiety affect partitioning into lipid, and that no correlation existed between the propensity of a pheromone to partition into the lipid and its biological activity. [source] | |||||||||||||||||||