Effect of the Methodology on Circulating Peptides Determination and Consequences on Net Flux Measurements Across the Gastrointestinal Tract of Sheep

Two methodologies for the measurement of peptide amino acids (PAA) in blood were compared to evaluate their effects on the measurement of the net flux of peptides across the gastrointestinal tract of sheep. These methods consisted of a chemical deproteinization of blood samples with sulfosalicylic acid (1.6M, 0.1 ml for 1 ml of sample) or perchloric acid (1M, 1 ml for 1 ml of sample) followed by ultrafiltration through a 3,000-Da cut-off filter (SSA+UF3kD) or gel filtration through a Sephadex G-15 column (1,500-Da cut-off filter; PCA+G-15), respectively, prior to PAA analysis. Peptide concentrations as determined by amino acid concentrations before and after hydrolysis of samples were slightly greater with the SSA+UF3kD (991 µM) than with the PCA+G-15 (605 µM) methodology. However, both methodologies gave similar net portal-drained viscera flux data in sheep fed on alfalfa pellets with histidine as the only significant uptake of peptide amino acid.     

Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicylic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde—3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.     

Rainbow trout (Oncorhynchus mykiss) recombinant IL-1beta and derived peptides induce migration of head-kidney leucocytes in vitro.

The present work provides the first information concerning the chemoattractant activity of trout recombinant IL-1beta and its derived peptides, referred to as P1, P2 and P3. The predicted rainbow trout mature interleukin-1beta peptide was produced as a recombinant protein in Escherichia coli. The first peptide, P1, corresponded to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and had an MW of 137 kDa. It was equivalent to a region known to be part of the receptor binding domain from the mammalian crystal structure of IL-1beta complexed to its receptor. P2 was used as control peptide, consisting of the same 12 amino acids as P1, but arranged in a random sequence (VVEEYIRAPPTT). P3 was synthesised to complex with an adjacent region of the IL-1 receptor, and corresponded to fragment 207-216 (YRRNTGVDIS) of the trout sequence, with an MW of 1.18 kDa. Migration was stimulated when leucocytes were exposed to concentrations of > or = 10 ng ml(-1) rIL-1beta. Peptide P3 also induced leucocyte migration, with an optimal dose of 0.25 mM being recorded. While P1 had no effect on cell migration when used alone, synergism was evident as a consequence of combining P1 with a suboptimal dose (0.01 mM) of P3. No synergism occurred when cells were exposed to a combination of P3 and the control peptide P2.     

Measurement of alpha-keto acids in plasma using an amino acid analyzer

A method for measuring keto acid concentrations in physiological fluids using an amino acid analyzer was developed. After preliminary deproteinization and removal of amino acids, reduction with sodium cyanoborohydride at 105 degrees C resulted in efficient conversion of the keto acids to their corresponding amino acids. In applying the technique to plasma samples, the use of MeOH for deproteinization was necessary to avoid the large losses of keto acids that occurred during precipitation of proteins with perchloric acid. The method was used to follow plasma ketoisocaproate concentrations in rat plasma after administration of leucine, and was sufficiently sensitive to detect concomitant changes in other branched-chain keto acid concentrations.     

Cholic acid modified 2 kDa polyethylenimine as efficient transfection agent

New lipopolymers were synthesized by conjugating cholic acid (ChA) to polyethylenimines (PEI; 2 and 25 kDa) and a polyallylamine (PAA; 15 kDa) via N‐acylation to develop effective gene delivery systems. The extent of ChA substitution linearly varied with the feed ratio during synthesis, indicating good control over grafting ratio. While ChA did not affect binding to plasmid DNA (pDNA) for higher molecular weight (MW) polymers, ChA substitution to 2 kDa PEI significantly affected the pDNA binding. Toxicity of the 2 kDa PEI was unaffected by ChA substitution, but it was improved for the higher MW polymers. Using immortal 293T cells and primary cord blood‐derived mesenchymal stem cells, low MW (2 kDa) PEI was shown to display much better transfection efficiency as a result of ChA substitution, unlike the higher MW polymers. We conclude that ChA could be a suitable substituent for non‐toxic (low MW) PEIs in order to improve their transfection efficiency. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1337–1341, 2013     

液质联用多反应监测法定量目标多肽或蛋白质

为建立优化的血浆内源性多肽提取方法,并且构建目标多肽和蛋白质的质谱定量方 法,本研究考察了超滤法、有机溶剂沉淀法和固相萃取法对血浆内源性多肽的提取效果 ,并通过Tricine-SDS-PAGE对提取效果进行比较.通过液相色谱串联质谱多反应监测 （MRM）分析,建立了多肽标准品ESAT-6定量方法,并将ESAT-6定量建立的液相色谱和质谱条件应用于蛋白质的定量,对多肽和蛋白质MRM定量的标准曲线进行了考 察.Tricine-SDS-PAGE结果表明,乙腈沉淀法是最佳的血浆内源性多肽提取方法,低分子量的多肽可以得到很好的富集,且能有效地去除高分子蛋白质的污染.液相色谱串联 质谱MRM法检测血浆内提取的多肽,标准曲线的线性较好,相关系数为0.999.另外,采 用MRM法对胶内分离的蛋白质进行定量,标准曲线的线性相关系数为0.995.综上所述, 本研究构建了一种简单有效的血浆多肽提取方法,通过液质联用MRM法成功地实现了目标多肽和蛋白质定量测定.该定量方法可以推广应用于复杂样品中的多肽和蛋白质的定 量分析.     

New approach for amino acid profiling in human plasma by selective fluorescence derivatization

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.     

Phenylalanine and its metabolites induce embryopathies in mouse embryos in culture

The aim of this study was to determine the teratogenicity of phenylalanine (Phe) and Phe metabolites in neurulating mouse embryos. Therefore, the system of whole embryo culture was employed and D9 (neurulating) mouse embryos were exposed to Phe, phenylethylamine (PEA), phenylpyruvic acid (PPA), phenylacetic acid (PAA), 2-OH phenylacetic acid (2-OH PAA), and phenyl-lactic acid (PLA) at concentrations ranging from 0.01 mM to 10 mM for 24 hours. After 24 hours, embryos were examined for morphological abnormalities and protein content by the Lowry method. Phe at 1 and 6 mM concentrations was not teratogenic; however, 10 mM inhibited cranial neural tube closure in 82% of the embryos. PEA was the most toxic factor and concentrations of 1 and 10 mM were embryo-lethal, whereas neural tube closure defects (NTDs) were observed in 67% of the embryos at 0.1 mM. 2-OH PAA was the second most toxic metabolite with concentrations of 1 and 10 mM producing NTDs in 10 and 100% of the embryos, respectively. PLA and PAA produced no NTDs at concentrations of 1 mM, 60% at 5 mM, and 100% at 10 mM. Finally, PPA produced approximately 50% NTDs at both 1 mM and 10 mM concentrations. PLA, PAA, 2-OH PAA, and PPA produced a significant reduction in embryonic protein, and PEA and 2-OH PAA reduced yolk sac protein values. PEA, 2-OH PAA, PPA, PAA, and PLA also produced craniofacial abnormalities, i.e., incomplete expansion of the forebrain, collapse of the optic vesicle, and hypoplasia of the mandible and/or the maxilla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.     

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

BMI and waist circumference in predicting cardiovascular risk factor clustering in Chinese adolescents

Objective: To derive the optimal BMI and waist circumference (WC) cut‐off values to predict clustering of cardiovascular risk factors in Hong Kong Chinese adolescents. Research Methods and Procedures: A total of 2102 Hong Kong Chinese 12 to 19 years of age were recruited. Participants were considered to have clustering of risk factors if at least three of the following risk factors were present: 1) high‐density lipoprotein cholesterol (HDL‐C) ≤1.03 mM, 2) low‐density lipoprotein cholesterol (LDL‐C) ≥2.6 mM, 3) triglyceride (TG) ≥1.24 mM, 4) fasting plasma glucose (FPG) ≥6.1 mM, and 5) age‐, sex‐, and height‐adjusted systolic or diastolic blood pressure (BP) ≥ 90th percentile. Receiver operating characteristics (ROC) curves were generated to identify the optimal age‐adjusted BMI and WC cut‐off values to predict clustering of risk factors in boys and girls separately. These age‐adjusted BMI and WC cut‐offs were transformed to percentile values. Cole's lambda‐mu‐sigma (LMS) method was used to obtain smoothed age‐specific BMI and WC at these percentile values. Results: The areas under ROC curves for BMI in girls and boys were 0.85 [95% confidence interval (CI), 0.77 to 0.92] and 0.76 (95% CI, 0.66 to 0.85), respectively. The respective areas under ROC curves for WC in girls and boys were 0.82 (95% CI, 0.74 to 0.91) and 0.78 (95% CI, 0.68 to 0.87). The optimal BMI thresholds were at the 78th percentile for girls and the 72nd percentile for boys. The respective values for WC were at the 77th percentile for girls and the 76th percentile for boys. The sensitivities and specificities of these cut‐off values ranged from 72% to 80%. Discussion: Age‐ and sex‐specific BMI and WC cut‐off values can be used to identify adolescents with clustering of cardiovascular risk factors.     

Microwave‐assisted TFA cleavage of peptides from Merrifield resin

Microwave‐assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off‐resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC‐MS and MS/MS experiments. In a 5 min microwave‐assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW‐assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI‐MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A novel defensin‐like peptide from salivary glands of the hard tick,Haemaphysalis longicornis

A novel defensin‐like antimicrobial peptide named longicornsin was isolated from the salivary glands of the hard tick, Haemaphysalis longicornis, using a 10‐kDa cut‐off Centriprep filter and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Its amino acid sequence was determined as DFGCGQGMIFMCQRRCMRLYPGSTGFCRGFRCMCDTHIPLRPPFMVG by Edman degradation. The cDNA encoding longicornsin was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 78 amino acids including a mature longicornsin. It showed similarity with defensin‐like peptides from other ticks by BLAST search. Different from most other tick defensin‐like peptides, longicornsin had a C‐terminal extension. Purified longicornsin exerted potent antimicrobial activities against bacteria and fungi. Interestingly, it even showed strong antimicrobial ability against drug‐resistant microorganisms and Helicobacter pylori. The results of this study indicated that longicornsin is a potential candidate for novel antimicrobial drug design.     

Enzymatic characterisation of the multifunctional enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-alpha-aminoadipic-acid-dependent ATP/PPi exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with alpha-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.     

Isolation and some properties of bovine brain 100 kDa heat shock protein

1. The 100 kDa protein was purified from bovine brains. 2. The antibody against the 100 kDa brain protein was prepared and was monospecific to the antigen. 3. The antibody cross-reacted with HeLa cell HSP100 (100 kDa heat shock protein). 4. The physicochemical, immunochemical properties and a partially amino acid sequence indicated that the 100 kDa protein was HSP100. 5. Peptide mapping using Staphylococcus aureus V8 protease showed a core peptide with 10 kDa molecular mass common to both HSP100 and HSP90. 6. The amino acid sequence of the 10 kDa fragment of the 100 kDa protein showed a high homology with that of human HSP90 (38-60); the difference was only two of 23 amino acid residues determined.     

A convenient microwave‐enhanced solid‐phase synthesis of short chain N‐methyl‐rich peptides

Structural modification of the peptide backbone via N‐methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)‐assisted Fmoc/tBu solid‐phase method to prepare short chain N‐methyl‐rich peptides, using Rink amide p‐methylbenzhydrylamine (MBHA) resin as solid‐phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc‐N‐methyl amino acid coupling. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Development and clinical application of an enzyme immunoassay for the determination of midregional proadrenomedullin

Adrenomedullin (ADM) is a 52‐amino acid peptide with a variety of physiologic functions such as immunomodulating activity, direct bactericidal activity, maintenance of renal homeostasis, and vasodilatory activity. Midregional proADM (MR‐proADM) is derived from a larger 185‐amino acid precursor peptide, prepro‐adrenomedulin (preproADM), by posttranslational processing. It is suggested to be co‐synthesized with ADM in equimolar amounts and has the advantages over ADM in having a longer half‐life, no bioactivity, and no binding to protein. Therefore, MR‐proADM serves as a surrogate for ADM secretion. In this study, we attempted to develop an enzyme immunoassay (EIA) for quantifying MR‐proADM‐like immunoreactive substance (IS), which is applicable for monitoring plasma MR‐proADM levels. By using β‐d ‐galactosidase‐labeled preproADM(83‐94) as a marker antigen, anti‐rabbit IgG‐coated immunoplate as a bound/free separator, and 4‐methylumbelliferyl‐β‐d ‐galactopyranoside as a fluorogenic substrate, a sensitive and specific EIA was developed for the quantification of MR‐proADM‐IS in human plasma. The lower limit of quantification was 0.032 pmol/well, and the steep competitive inhibition EIA calibration curve obtained was linear between 0.16 and 10 nmol/L. By using human plasma samples containing 0.2 and 2.0 nmol/L of MR‐proADM, the interassay coefficients of variation (reproducibility) were 10.78% and 8.83%, respectively, and intraassay coefficients were 3.91% and 7.81%. Plasma MR‐proADM‐IS level was significantly higher in patients with chronic renal failure (1.39 ± 0.50 nmol/L) compared with healthy subjects (0.19 ± 0.07 nmol/L). These results suggest that our EIA may be useful to evaluate plasma MR‐proADM levels as a biomarker in various clinical settings. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Developmental changes of amino acids in ovine fetal fluids

We recently reported an unusual abundance of arginine (4-6 mM) in porcine allantoic fluid during early gestation. However, it is not known whether such high concentrations of arginine are unique for porcine allantoic fluid or whether they represent an important physiological phenomenon for mammals. The present study was conducted to test the hypothesis that arginine is also the most abundant amino acid in ovine allantoic fluid. Allantoic and amniotic fluids, as well as fetal and maternal plasma samples, were obtained from ewes between Days 30 and 140 of gestation. Glycine was the most abundant amino acid in maternal uterine arterial plasma, representing approximately 25% of total alpha-amino acids. Alanine, glutamine, glycine, plus serine contributed approximately 50% of total alpha-amino acids in fetal plasma. Fetal:maternal plasma ratios for amino acids varied greatly, being less than 1 for glutamate during late gestation, 1.5-3 for most amino acids throughout gestation, and greater than 10 for serine during late gestation. Marked changes were observed in amino acid concentrations in amniotic and allantoic fluids associated with conceptus development. Concentrations of alanine, citrulline, and glutamine in allantoic fluid increased by 20-, 34-, and 18-fold, respectively, between Days 30 and 60 of gestation and were 24.7, 9.7, and 23.5 mM, respectively, on Day 60 of gestation (compared with 0.8 mM arginine). Remarkably, alanine, citrulline, plus glutamine accounted for approximately 80% of total alpha-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed approximately 60% of total alpha-amino acids in allantoic fluid on Day 140 of gestation. These novel findings of the unusual abundance of traditionally classified nonessential amino acids in allantoic fluid raise important questions regarding their roles in ovine conceptus development.     

Quinine‐Based Zwitterionic Chiral Stationary Phase as a Complementary Tool for Peptide Analysis: Mobile Phase Effects on Enantio‐ and Stereoselectivity of Underivatized Oligopeptides

Peptide stereoisomer analysis is of importance for quality control of therapeutic peptides, the analysis of stereochemical integrity of bioactive peptides in food, and the elucidation of the stereochemistry of peptides from a natural chiral pool which often contains one or more D‐amino acid residues. In this work, a series of model peptide stereoisomers (enantiomers and diastereomers) were analyzed on a zwitterionic ion‐exchanger chiral stationary phase (Chiralpak ZWIX(+) 5 µm), in order to investigate the retention and separation performance for such compounds on this chiral stationary phase and elucidate its utility for this purpose. The goal of the study focused on 1) investigations of the effects of the sample matrix used to dissolve the peptide samples; 2) optimization of the mobile phase (enabling deriving information on factors of relevance for retention and separation); and 3) derivation of structure–selectivity relationships. It turned out that small di‐ and tripeptides can be well resolved under optimized conditions, typically with resolutions larger than 1.5. The optimized mobile phase often consisted of methanol–tetrahydrofuran–water (49:49:2; v/v/v) with 25 mM formic acid and 12.5 mM diethylamine. This work proposes some guidance on which mobile phases can be most efficiently used for peptide stereoisomer separations on Chiralpak ZWIX. Chirality 28:5–16, 2016. © 2015 Wiley Periodicals, Inc.     

A simple method for determination of intramolecular amino acid sequence of unpurified protein. Application to human serum protein adsorbed by silica particles.

Silica particles adsorbed several kinds of human serum proteins, especially 23 kDa molecular weight protein. After SDS-PAGE of adsorbed serum proteins, gel pieces containing 23 kDa protein was cut out and set in slot of stacking gel in second SDS-PAGE following overlay of Staphylococcus aureus V8 protease. After electrophoresis, gel was subjected to electroblotting onto polyvinylidene difluoride membrane. Both bands of dye-stained 23 kDa and the peptide were cut out from membrane and analyzed for amino acid sequence. Obtained sequences agreed well with amino terminal and intramolecular sequences of human HDL-apolipoprotein, A-I.