Influence of an Unnatural Amino Acid Side Chain on the Conformational Dynamics of Peptides

In this work, a non‐natural amino acid, H‐propargylglycine‐OH (Pra), is chosen to examine the side‐chain effect on the backbone conformation of small peptides. The conformations of two synthesized Pra‐containing tripeptides, Ac‐Pra‐Pra‐NH₂ (PPTP) and Ac‐Pra‐Ala‐NH₂ (PATP), are examined by infrared (IR) spectroscopy in combination with molecular dynamics (MD) simulations and quantum chemical computations. By analyzing the joint distributions of backbone torsional angles, several significant conformations can be identified for the two tripeptides solvated in D₂O. At room temperature, 44 % of PPTP exists in the α‐α conformation and 33 % of PATP exists in the α‐polyproline‐II conformation. Larger structural inhomogeneity is seen in both cases by MD simulations at elevated temperatures. Thus even a small side chain, such as the propargyl group can significantly alter the peptide backbone conformations. The results suggest that there is no overwhelming conformational propensity of the Pra residue in short peptides. IR spectra simulated in the amide‐I region using two different methods, reasonably reproduce the experimental IR spectra and their temperature dependence.     

Examining the Influence of Phosphorylation on Peptide Ion Structure by Ion Mobility Spectrometry-Mass Spectrometry

Ion mobility spectrometry-mass spectrometry (IMS-MS) techniques are used to study the general effects of phosphorylation on peptide structure. Cross sections for a library of 66 singly phosphorylated peptide ions from 33 pairs of positional isomers, and unmodified analogues were measured. Intrinsic size parameters (ISPs) derived from these measurements yield calculated collision cross sections for 85% of these phosphopeptide sequences that are within ±2.5% of experimental values. The average ISP for the phosphoryl group (0.64 ± 0.05) suggests that in general this moiety forms intramolecular interactions with the neighboring residues and peptide backbone, resulting in relatively compact structures. We assess the capability of ion mobility to separate positional isomers (i.e., peptide sequences that differ only in the location of the modification) and find that more than half of the isomeric pairs have >1% difference in collision cross section. Phosphorylation is also found to influence populations of structures that differ in the cis/trans orientation of Xaa–Pro peptide bonds. Several sequences with phosphorylated Ser or Thr residues located N-terminally adjacent to Pro residues show fewer conformations compared to the unmodified sequences.

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Buffer Influence on the Amino Acid Silica Interaction

Protein-surface interactions are exploited in various processes in life sciences and biotechnology. Many of such processes are performed in presence of a buffer system, which is generally believed to have an influence on the protein-surface interaction but is rarely investigated systematically. Combining experimental and theoretical methodologies, we herein demonstrate the strong influence of the buffer type on protein-surface interactions. Using state of the art chromatographic experiments, we measure the interaction between individual amino acids and silica, as a reference to understand protein-surface interactions. Among all the 20 proteinogenic amino acids studied, we found that arginine (R) and lysine (K) bind most strongly to silica, a finding validated by free energy calculations. We further measured the binding of R and K at different pH in presence of two different buffers, MOPS (3-(N-morpholino)propanesulfonic acid) and TRIS (tris(hydroxymethyl)aminomethane), and find dramatically different behavior. In presence of TRIS, the binding affinity of R/K increases with pH, whereas we observe an opposite trend for MOPS. These results can be understood using a multiscale modelling framework combining molecular dynamics simulation and Langmuir adsorption model. The modelling approach helps to optimize buffer conditions in various fields like biosensors, drug delivery or bio separation engineering prior to the experiment.     

蛋白质水解阶段对氨基酸组成分析的影响

水解是蛋白质氨基酸组成分析的重要步骤，水解质量的好坏直接影响到分析结果的正确与否，蛋白质的水解受到很多因素的影响，如温度，时间、水解试剂，添加剂，水解方法等，本文对近10年来蛋白质组成分析中中有关蛋白质水解问题的研究进进行了评述，对最近几年出现的一些新的水解方法如酸性水解中的气相水解，微波辐射水解，膜上蛋白质和印迹水解，聚丙烯酰安凝胶中蛋白质的水解，敏感氨基酸的水解以及水解过程中影响氨基酸外消旋化的因素等作了简要的介绍。     

Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

Previously we have shown that subcritical water may be used as an alternative to enzymatic digestion in the proteolysis of proteins for bottom-up proteomics. Subcritical water hydrolysis of proteins was shown to result in protein sequence coverages greater than or equal to that obtained following digestion with trypsin; however, the percentage of peptide spectral matches for the samples treated with trypsin were consistently greater than for those treated with subcritical water. This observation suggests that in addition to cleavage of the peptide bond, subcritical water treatment results in other hydrolysis products, possibly due to modifications of amino acid side chains. Here, a model peptide comprising all common amino acid residues (VQSIKCADFLHYMENPTWGR) and two further model peptides (VCFQYMDRGDR and VQSIKADFLHYENPTWGR) were treated with subcritical water with the aim of probing any induced amino acid side-chain modifications. The hydrolysis products were analyzed by direct infusion electrospray tandem mass spectrometry, either collision-induced dissociation or electron transfer dissociation, and liquid chromatography collision-induced dissociation tandem mass spectrometry. The results show preferential oxidation of cysteine to sulfinic and sulfonic acid, and oxidation of methionine. In the absence of cysteine and methionine, oxidation of tryptophan was observed. In addition, water loss from aspartic acid and C-terminal amidation were observed in harsher subcritical water conditions.

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On the Preparation of Enantiomerically Pure Isonitriles from Amino Acid Esters and Peptides

An improved method for the synthesis of enantiomerically pure isonitriles from amino acid esters and dipeptides is described.     

Ion Exchanger Liquid Chromatography of N-Acetylaspartic Acid and Some N-Acetylaspartyl Peptides

Abstract

A high performance liquid chromatographic (HPLC) procedure for the rapid separation of N-acetylaspartic acid, N-acetyl-aspartyl-glutamic acid and N-acetylaspartyl-glutamyl-aspartic acid is described. The procedure utilizes a pellicular ion-exchange column support, and ionic strength gradient with mobile phase solutions buffered to pH 5.0 and a UV detector operated at 210 nm. Reproducibility and quantitative capabilities are also discussed. The method has been used for a tentative estimation of N-acetylaspartic acid and N-acetylaspartyl peptides in a rat brain synaptosomal extract.     

High-Mass MALDI-MS Using Ion Conversion Dynode Detectors: Influence of the Conversion Voltage on Sensitivity and Spectral Quality

With the development of special ion conversion dynode (ICD) detectors for high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the mass-to-charge ratio is no longer a limiting factor. Although these detectors have been successfully used in the past, there is lack of understanding of the basic processes in the detector. We present a systematic study to investigate the performance of such an ICD detector and separate the contributions of the MALDI process from the ones of the ion-to-secondary ion and the secondary ion-to-electron conversions. The performance was evaluated as a function of the voltages applied to the conversion dynodes and the sample amount utilized, and we found that the detector reflects the MALDI process correctly: limitations such as sensitivity or deviations from the expected signal intensity ratios originate from the MALDI process itself and not from the detector.

Study on the Enhancement of Proton Affinity by N-Diisopropyloxy Phosphorylation of Amino Acid in Mass Spectrometry

With introduction of a diisopropyloxy phosphoryl group into the N terminal of amino acids,it was found that proton affinity(PA) of amino acid was enhanced in mass spectrometry.Density functional theory calculations showed that the energy for protonation of DIPP-amino acid is lower than that of amino acid,which means PA of DIPP-AA ishigher than that of corresponding amino acid.These results.Coincident with our empirical results.offer a useful interpretation of experimental observations.     

Effect of Cysteic Acid Position on the Negative Ion Fragmentation of Proteolytic Derived Peptides

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M – H]⁻ ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon–carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C_oxLRRASLG) [M – H + O₃]⁻ ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d _i fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a _i ions followed by the elimination of the side chain to form d _i ions).     

人参中氨基酸及多肽的研究

氨基酸及多肽是人参中重要成分之一。田中治,Gstirner等对人参根中氨基酸分析表明,主要有Arg、Lys、Ala、Ser、Tyr、Pro、Gly、Glu、Thr、Leu、Ile、Val、AspPhe、His等,也曾确定含半胱氨酸。我们分析了吉林省集安边条参各部位的氨基酸(见表1)表明,芦头和花蕾各检出16种;根检出15种;侧根、须根和叶各检出14种。     

离子色谱间接抑制电导检测氨基酸类物质

间接色谱法是一种色谱检测的新技术 ,它采用对检测器是活性的流动相 ,使之形成一个常数信号 ,当非活性的离子交换组份通过检测器时 ,信号减小 ,从而可间接地测定该组分。间接色谱法已经被辐射、光度、安培、电导、荧光等检测法所采用 ,但尚未有过间接抑制电导的报道。作者利用离子色谱间接抑制电导检测法测定了氨基酸类物质 ,结果较为满意     

氨基酸、多肽的环糊精化学

本文着重介绍了氨基酸、多肽－环糊精连接物的合成,分子识别和自组装,对环糊精及其衍生物与氨基酸、多肽的包合行为,异构体识别和仿酶合成作了简要概述。     

The Impact of Amino Acid Side Chain Mutations in Conformational Design of Peptides and Proteins

Local energetic effects of amino acid replacements are often considered to have only a moderate influence on the backbone conformation of proteins or peptides. As these effects are difficult to determine experimentally, no comparison has yet been performed. However, knowledge of the influence of side chain mutations is essential in protein homology modeling and in optimizing biologically active peptide ligands in medicinal chemistry. Furthermore, the tool of N‐methylation of peptides is of increasing importance for the design of peptidic drugs to gain oral availability or receptor selectivity. However, N‐methylation is often accompanied by considerable population of cis‐peptide bond structures, resulting in completely different conformations compared with the parent peptide. To retain a favored structure, it might be important to understand the effect of different side chains on the backbone conformation and to enable the introduction of an N‐methylation at the right position without disturbing a biologically active conformation. In order to detect even small energetic effects due to side chain mutations, we employed a trick to investigate the structural equilibrium of a selected cyclic pentapeptide in which two conformations are equally populated. Very small energetic differences between both conformations could easily be determined experimentally by identifying shifts in the population of both isomers.     

Isolation and Amino Acid Composition of Human Milk Casein and its Components

A modified procedure is described for isolation of whole human milk casein. Human milk casein has been separated into three fractions. The gamma fraction was found to be more abundant than the beta-fraction, while the alpha-fraction occurred only in tracer amount. The amino acid composition, electrophoretic mobilities, and the side chain groups of the human milk casein and its fractions have been determined.     

Cathodic Stripping Voltammetry of Sulphur-Containing Amino Acids and Peptides in the Presence of Nickel Ion. Catalytic and Inhibiting Effects

Abstract

The paper reviews recent studies on the effect of addition of Ni(II) in the cathodic stripping voltammetry of the following compounds: cysteine, penicillamine, cystine, glutathione (either reduced or oxidised) and N-acetylcysteine. With the exception of N-acetylcysteine, the above compounds give a cathodic stripping peak at -0.6 V (vs. Ag/AgCl, 3 M KCl electrode) which is due to the catalytic reduction of nickel ion. Even in the case of the disulphides the actual catalyst is the thiol produced by the cleavage of the -S-S- bond during the accumulation step. The catalytic peak enables the detection of the analyte with a better selectivity than is obtained with the stripping peak due to the reduction of mercury thiolates. In addition, Ni(II) suppresses the mercury thiolate peak of ligands such as cysteine or penicillamine, but does not modify the behaviour of thiols with low complexing properties (such as N-acetylcysteine). Consequently, compounds such as cysteine and its N-acyl derivatives can be determined simultaneously by means of the catalytic peak and the mercury thiolate peak (at -0.4 V) respectively.     

Production,Purification, and Study of the Amino Acid Composition of Microalgae Proteins

Microalgae are known to be rich in protein. In this study, we aim to investigate methods of producing and purifying proteins of 98 microalgae including Chlorella vulgaris, Arthrospira platensis, Nostoc sp., Dunaliella salina, and Pleurochrysis carterae (Baltic Sea). Therefore, we studied their amino acid composition and developed a two-stage protein concentrate purification method from the microalgae biomass. After an additional stage of purification, the mass fraction of protein substances with a molecular weight greater than 50 kDa in the protein concentrate isolated from the biomass of the microalga Dunaliella salina increased by 2.58 times as compared with the mass fraction before filtration. In the protein concentrate isolated from the biomass of the microalga Pleurochrysis cartera, the relative content of the fraction with a molecular weight greater than 50.0 kDa reached 82.4%, which was 2.43 times higher than the relative content of the same fractions in the protein concentrate isolated from this culture before the two-stage purification. The possibilities of large-scale industrial production of microalgae biomass and an expanded range of uses determine the need to search for highly productive protein strains of microalgae and to optimize the conditions for isolating amino acids from them.