GC-MS identification of proteins in wall painting samples: A fast clean-up procedure to remove copper-based pigment interferences

A new approach was explored to purify proteins in a multi-step procedure for the characterisation of proteinaceous materials (casein, animal glue, and egg) in artwork samples by gas chromatography-mass spectrometry. High concentrations of inorganic salts, such as azurite, have been found to impair the determination of protein via amino acid analysis. The effect of varying concentrations of copper-based pigments on the quantification of amino acids was evaluated through the analysis of replica paintings prepared with the three types of proteinaceous materials. Glycine, aspartic and glutamic acids are the amino acids most affected by the presence of copper salts. In the case of high concentration of salts, this interference hampers the correct identification of the proteins. To eliminate the inorganic salts, a C18 pipette tip was used to clean-up the ammonia extracts before the acidic hydrolysis step. The clean-up procedure allows us to prevent the influence of the inorganic salts and thus allows correct protein identification, though the quantitative recovery of proteinaceous material is quite low. The effectiveness of the optimised procedure was evaluated by analysing samples from two Italian wall paintings from the 13th and the 14th centuries. Without the clean-up it would not have been possible to detect the presence of a mixture of egg and animal glue in one case, and that of egg in the other one.     

Application of the Kohonen artificial neural network in the identification of proteinaceous binders in samples of panel painting using gas chromatography-mass spectrometry

Historically, three types of proteinaceous matter--casein, egg and animal glue--were used as binders for pigments or as adhesives in easel and wall painting. The relative percentage content of alanine, glycine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, aspartic acid, glutamic acid, lysine, methionine, proline and hydroxyproline, as determined by GC-MS, is used for binder identification. In this paper we analyse the viability of a multivariate modelling using Kohonen's neural network to characterise the wood adhesive in 16 old samples from Italian panel paintings of the 12-16th centuries. As a training set we use the amino acid composition of 141 samples contributed by the Opificio delle Pietre Dure of Florence (Cultural Heritage Ministry, Italy). Of the 141 samples, 113 were used to train the Kohonen neural network and the remaining 28 as the evaluation set. A specificity and sensitivity of 100% was achieved in training and 92-100% in prediction depending on the assignation criteria employed. The neural network thus trained and evaluated was applied to the old samples, achieving identification of all of them. In addition, the map obtained for each amino acid provides relevant information as to its importance in the characterisation of the sample.     

基于化学模式识别的大蒜傅里叶变换红外光谱指纹图谱多样性

以不同地理居群的大蒜为研究对象,采用傅里叶变换红外光谱法获取各样品的红外谱图及不同波数下各样品的透过率数据,运用主成分分析及聚类分析等统计学方法对谱图数据进行了比较分析。结果发现,不同地理居群大蒜的红外指纹图谱存在差异,统计学分析结果显示25个品种大致可分为4大类(其中2个样品,4号及25号与这4类间有较大差异),此现象一定程度上反映了大蒜种植地居群环境对大蒜理化性质的影响。     

Direct amino acid analysis method for speciation of selenoamino acids using high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection

Speciation analysis of selenomethylcysteine (SeMeCys), selenomethionine (SeMet) and selenocystine (SeCys) has been performed using a direct amino acid analysis method with high-performance anion-exchange chromatography (HPAEC) coupled with integrated pulsed amperometric detection (IPAD). Three selenoamino acids could be baseline-separated from 19 amino acids using gradient elution conditions for amino acids and determined under new six-potential waveform. Detection limits for SeMeCys, SeMet and SeCys were 0.25, 1 and 20 microg/L (25 microL injection, 10 times of the baseline noise), respectively. The relative standard deviations (RSDs) of 200 microg/L SeMeCys, SeMet and SeCys were 3.1, 4.1 and 2.8%, respectively (n=9, 25 microL injection). The proposed method has been applied for determination of selenoamino acids in extracts of garlic and selenious yeast granule samples. No selenoamino acids were found in garlic. Both SeMet and SeCys were detected in selenious yeast tablet with the content of 45 and 129 microg Se/g, respectively. Selenoamino acids standards were spiked in garlic and yeast granule samples and the recovery ranged from 90 to 106%.     

Identification of proteinaceous binding media of easel paintings by gas chromatography of the amino acid derivatives after catalytic hydrolysis by a protonated cation exchanger

Summary A procedure for the hydrolysis of proteins, based on catalysis by the protonated form of a strong cation exchanger, was established for proteinaceous binding media (e.g. casein, egg, animal collagene) used for objects of art. The experimental parameters for the hydrolysis (temperature, time) were optimized, as well as the conditions for sorption and desorption of the amino acids on the cation exchanger. The results for the identification of proteins by the gas chromatographic pattern of the amino acid derivatives, after hydrolysis by the ion exchanger and by hydrochloric acid were compared. The former method proved to be more efficient due to the mild conditions, avoiding the formation of humins in the presence of carbohydrates, and reducing the dissolution of pigments. The method was applied to identify the proteinaceous vehicles of samples from priming and paint layers of easel and wall paintings from the 16th and 18th century.     

A peptide containing aspartic acid, glutamic acid and serine in calf brain synaptic vesicles

Free amino acids and other amino compounds in calf brain synaptic vesicles were identified and determined by thin-layer chromatography and ion-exchange chromatography. The vesicles contained ten identified amino acids with glutamic acid, aspartic acid, taurine and gamma-aminobutyric acid in the highest concentrations, and also cysteic acid (or cysteinesulfinic acid), glutamine, alanine, serine, glycine and lysine. The vesicles also contained certain unknown acid-labile, ninhydrin-positive compounds, one of which was a peptide yielding, after acid hydrolysis, about 40% aspartic acid, 30% serine, 15% glutamic acid, 10% glycine and possibly some alanine and lysine. The concentration of the peptide in the vesicles was as high as that of all the other amino compounds together.     

超高效液相色谱-高分辨质谱法测定中华绒螯蟹中游离氨基酸

游离氨基酸不仅是一类重要的营养物质,更与中华绒螯蟹独特的滋味和香味密切相关。对游离氨基酸含量进行测定,能为中华绒螯蟹产品的品质评价和风味研究提供有价值的信息。该工作通过优选提取溶剂、优化仪器测定参数,最终使用5%(v/v)高氯酸水溶液提取目标物,采用XDB-C₁₈色谱柱(100 mm×4.6 mm, 1.7μm),以0.1%(v/v)甲酸水溶液-乙腈为流动相进行梯度洗脱,在电喷雾正离子电离、选择离子扫描模式下进行检测,外标法定量,建立了超高效液相色谱-高分辨质谱(UHPLC-HRMS)测定中华绒螯蟹中17种游离氨基酸含量的方法。结果表明,17种氨基酸在10.0～200.0 mg/L范围内线性关系良好,相关系数(R²)≥0.999 0,仪器检出限为0.3 mg/L,仪器定量限为1.0 mg/L。在中华绒螯蟹可食部分中按三水平进行加标回收试验,各目标物的回收率在78.4%～105.3%之间。通过对仪器和方法精密度进行评估,发现17种氨基酸测定仪器重复性的RSD≤4.2%,方法重复性试验的RSD≤5.2%,再现性试验的RSD≤11.4%,精密度数据...     

Use of high performance liquid chromatography in defining the abnormalities in the free amino acid patterns in the cerebrospinal fluid of patients with aseptic meningitis.

Free amino acids were quantitatively determined in cerebrospinal fluid (CSF) and plasma samples from patients with aseptic meningitis by a newly developed high performance liquid chromatographic (HPLC) method. The method of analysis was based on precolumn derivatization of orthophthaladehyde in the presence of 2-mercaptoethanol and detection was made at Eex = 340 nm and Eem = 450 nm. The method was sensitive and the limit for detection was less than 1 pmol for most of the amino acids. It took 45 min to separate 26 amino acids with highly reproducible results, giving a coefficient of variance for retention times and integrated areas less than 0.4% and 2%, respectively, after five replicate runs. The results accumulated in 10 patients were compared statistically with 11 age-matched healthy controls. Among the amino acids almost all the neurotransmitter candidates, such as aspartic acid, glutamic acid, glutamine, glycine, tyrosine, phenylalanine and gamma-aminobutyric acid (GABA), were significantly increased in the patients' CSF, whereas arginine and threonine were low. No change was observed in plasma amino acids in patients as compared to healthy controls. The higher levels of most of the neurotransmitters, especially GABA, aspartic acid and glutamic acid, could be used diagnostically in assessing the progression and remission in aseptic meningitis.     

Investigation of contemporary gilded forgeries of ancient coins

Four contemporary forgeries of ancient gold coins were investigated regarding techniques used for gilding, and the composition of the gold cover and the base metal core. The forged coins are a Daric of the Persian Empire, a Gold Stater in the name of Alexander, and two Solidi of the late Roman Empire. A combination of modern analytical methods such as Scanning Electron Microscopy (SEM), Electron Probe Micro Analysis (EPMA), X-Ray Fluorescence Spectrometry (XRF), and SecondaryIon Mass Spectrometry (SIMS) was used. The results demonstrate that the coins represent the main three technologies of gilding used in antiquity. The core of the Daric is a silver Siglos, plated by leaf gilding. The Gold Stater was forged by foil gilding using a silver core. The Roman Solidi have a core of either silver or copper and were plated by fire gilding. On account of our results it is possible to compare the forgers' profits made by use of the different technologies of forging.     

The changes of fatty acid and amino acid compositions in sea bream (Sparus aurata) during irradiation process

Aqua cultured fish (sea bream) were irradiated by Cobalt-60 at commercial irradiation facility at dose of 2.5 and 5 kGy at 2–4 °C. The proximate composition, fatty acid and amino acid composition changes of irradiated aqua cultured sea bream (Sparus aurata) of Aegean Sea were investigated. Total saturated (28.01%) and total monounsaturated (28.42%) fatty acid contents of non-irradiated decreased content of 27.69–27.97% for 2.5 kGy irradiated groups and increased content of 28.33–28.56% for 5 kGy irradiated groups after irradiation process. Total polyunsaturated fatty acid content for irradiated samples was lower than that of non-irradiated samples. Aspartic acid, glutamic acid, serine, glycine, arginine, alanine, tyrosine, cystine, tryptophan, lysine and proline contents for 2.5 and 5 kGy irradiated sea bream are significantly different (p<0.05).     

Alterations of plasma and cerebrospinal fluid glutamate levels in rats treated with the N-methyl-D-aspartate receptor antagonist, ketamine

It has been reported that the repeated administration of a sub-anesthetic dose of an N-methyl-D-aspartate receptor antagonist, ketamine, can produce an animal model of schizophrenia. Since no information is available on the alterations of the amino acid levels in ketamine-treated rats, we investigated the amino acid composition in the plasma and cerebrospinal fluid of rats that were repeatedly administered with ketamine for 5 consecutive days (30 mg/kg/day). The plasma and cerebrospinal fluid amino acid compositions in the fifth week after cessation of repeated ketamine administration were determined by highperformance liquid chromatography with fluorescence detection using a pre-column fluorescence reagent, i.e. 4-fluoro-7nitro-2,1,3-benzoxadiazole. Among the amino acids investigated in the present study, the level of plasma glutamic acid increased significantly (p < 0.05), while that of the cerebrospinal fluid glutamic acid decreased significantly in the ketamine-treated rats as compared with these levels in control rats injected with saline (p < 0.05, n = 7). These alterations in the glutamic acid level in the plasma and cerebrospinal fluid resemble those in schizophrenic patients, suggesting that ketamine-treated rats may be a useful model for performing research on the pathophysiology of schizophrenia.     

柱前衍生-高效液相色谱法同时测定血清中氨基酸类及单胺类神经递质

以邻苯二甲醛（o-phthalaldehyde，OPA）为衍生试剂，建立了柱前衍生-高效液相色谱（HPLC）同时测定血清中氨基酸类神经递质牛磺酸（Tau）、谷氨酸（Glu）、甘氨酸（Gly）、γ-氨基丁酸（γ-GABA）和单胺类神经递质多巴胺（DA）含量的分析方法。血清与乙醇以1：2的体积比混合，进行蛋白质沉淀后离心，取其上清液，氮吹至近干。前处理后的样品与OPA进行柱前衍生，衍生化产物采用Luna 5u C18色谱柱（250 mm×4.6 mm，5 μm）分离，以柠檬酸-乙酸钠缓冲溶液（pH 3.73）为流动相A、乙腈为流动相B进行梯度洗脱，流速为1.0 mL/min，柱温为30℃，检测波长为338 nm。5种神经递质在各自范围内线性关系良好（r²≥0.9866），检出限为0.10~0.40 μmol/L，不同加标水平下目标物的加标回收率为87.57%~115.31%，相对标准偏差均低于7.80%。方法操作简单，灵敏度高，精密度、线性关系和回收率等方法学指标较好，可实现血清中氨基酸类及单胺类神经递质的同时检测。     

Thermoanalytical study of acid-treated clay containing amino acid immobilized on its surface

The aim of this study is to investigate the incorporation of amino acid molecules in an acid-activated montmorillonite by means of solid characterization after the incorporation of these biomolecules. The acid activation procedure was carried out for the purpose of increasing the acid sites in the clay as well as the impurity elimination in the mineral. Cysteine, aspartic, and glutamic acids were adsorbed on montmorillonite K10 which was previously treated with a hydrochloric acid solution. The clay was put in contact with amino acid solutions at two different concentrations. Each amino acid was adsorbed at identical conditions, with the pH fixed to ensure the charge of molecules and surface clay. The solid was characterized by means of X-ray diffraction, infrared spectroscopy, thermogravimetric analysis, and nitrogen adsorption at 77 K. After the amino acid adsorption, the powders showed changes in their characteristics as well as in their thermal behavior, which depended on both the concentration and the nature of the adsorbed amino acid. The thermal decomposition and elimination of cysteine occurred at a higher temperature than the aspartic and glutamic acid; the complete removal of glutamic acid molecules was not observed at 850 °C. The differences observed in the solid characteristic after the adsorption of each amino acid were discussed. Both the thermoanalytical study and characterization of materials after the interaction with amino acid molecules can be useful to understand the adsorption mechanism of biomolecules on solid surfaces.     

The development of a gas chromatographic–mass spectrometric analytical procedure for the determination of lipids,proteins and resins in the same paint micro-sample avoiding interferences from inorganic media

This paper presents a GC–MS analytical procedure for determining proteinaceous materials, glycerolipids, natural waxes and terpenoid resins in the same paint micro-sample. The procedure is also reliable when high amounts of interfering inorganic pigments, dryers and charges are present. The characterisation of proteinaceous binders in a paint sample can be subject to analytical interferences by inorganic materials. Such materials may form complexes with functional groups of proteins, thus preventing their efficient derivatisation, which is necessary prior to GC analysis. For this reason an analytical procedure has been developed based on two extractions and a clean-up step, in order to obtain two fractions: a lipid-resinous fraction and a proteinaceous fraction. The lipid-resinous fraction is subjected to salification/saponification assisted by microwaves, followed by acidification, extraction, derivatisation and GC–MS analysis. The proteinaceous fraction is analysed by GC–MS after hydrolysis and derivatisation of the freed amino acids. The desalting step is applied before the hydrolysis, and is based on the use of the monolithic sorbent tip technology with a C4 stationary phase. Reference paint replicas of egg, casein and animal glue were prepared with and without several metals containing pigments, and used to develop and validate the analytical procedure. The procedure proved to be efficient in desalting the proteinaceous materials both from cations and anions. Although non quantitative, it is reliable in the analysis of samples whose content of extractable proteins is <1 μg, thus showing it to be suitable for the characterisation of paint samples. An example of how the analytical procedure was used to characterise a sample from a 15th century panel painting is also discussed.     

Characterisation and identification of proteinaceous binding media (animal glues) from their amino acid profile by capillary zone electrophoresis

A capillary electrophoretic method for identifying different species of proteinaceous binders--collagen, egg white, and milk casein--is described. It allows characterisation of the proteins on the basis of the amino acid profiles obtained after their acidic hydrolysis. The profiles of the underivatised amino acids are recorded directly by capillary zone electrophoresis at pH 2.26 with the aid of a conductivity detector, thus eliminating the need for a derivatisation step. Identification is carried out by means of different relative peak areas of the amino acids. A scheme is given for identification, which is based on the main markers, hydroxyproline, proline, glycine, glutamic acid, and serine and valine.     

Metabolic profiling of amino acids in cellular samples via zwitterionic sub-2 μm particle size HILIC-MS/MS and a uniformly 13C labeled internal standard

A novel analytical method using hydrophilic interaction liquid chromatography combined with electrospray tandem mass spectrometry for metabolic profiling of free, underivatized amino acids is presented. The separation uses a zwitterionic modified silica-based stationary phase with 1.8-μm particle size functionalized with ammonium sulfonic acid groups. Quantification is based on external standard calibration using a Pichia pastoris cell extract grown on uniformly ¹³C labeled glucose as an internal standard. The absolute limits of detection in the cellular matrix were in the subpicomolar range. Measurement accuracy was assessed by analyzing NIST Standard Reference Material 2389a, which provides certified values for 17 amino acids. The recovery of the amino acids ranged between 65 % (proline) and 120 % (lysine), with excellent repeatability precision below 2.5 % (n?=?5). Only, cystine showed poor recovery (29 %) and repeatability precision (13 %). Generally, the long-term precision obtained by hydrophilic interaction liquid chromatography–tandem mass spectrometry was excellent, being on average less than 9 % over 20 h of measurement time. Moreover, the novel separation method had average repeatability and reproducibility of the chromatographic peak width over time periods of 20 h and 6 months of 8 and 15 %, respectively, demonstrating its high robustness in routine analysis of cellular samples. Large concentration differences depending on the amino acid were found in the cell extracts, typically ranging from 0.002 nmol per milligram of cell dry weight (cystine) to 56 nmol per milligram of cell dry weight (arginine and glutamic acid).     

Rapid method for the assay of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain tissue and subcellular fractions

The thin-layer electrophoretic separation at pH 4.8 of brain extracts and a procedure for fluorescent staining of the plates with fluorescamine are described for the rapid routine determination of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain extracts and in particulate fractions of brain tissue. Automated sample application, electrophoretic separation using two chambers, and quantitation by in situ fluorescence scanning allows the assay of 280 samples within three working days. The method is reproducible (S.D. less than 8% of the mean) within the range of 0.2--2 nmole per spot. The staining procedure can be applied to a variety of related analytical problems. The method has proved useful for the determination of the specific radioactivities of GABA, glutamic acid and aspartic acid in metabolic studies.     

谷氨酸在乳状液膜中的迁移行为

用氯化三等基·甲基铵（Ａｌｉｑｕａｔ ３３６）－Ｓｐａｎ８０－甲苯制成的乳状液膜体系对谷氨酸的迁移行为进行了研究,２ ｍｉｎ的迁移率可达９３％。在谷氨酸的最佳迁移条件下,油溶性好的苯丙氨酸迁移率较高,但油溶性低的甘氨酸和碱性氨基酸的迁移率明显较低。此法适用于微量氨基酸的提取和分离。     

Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells

A rapid, reproducible and highly sensitive method, based on liquid chromatography mass spectrometry, was developed for the determination of the excitatory amino acid glutamic acid released in the diffusion medium of control, ischemic and mutant cells from retinas. Signal intensity of glutamic acid was enhanced by dansyl chloride derivatization giving rise to a detection limit in the order of pmol/mL. Further, in HPLC-ESI-MS detection an MS-friendly dansyl group to glutamic acid enhanced both ionization efficiency in the ESI source and collision-activated dissociation in the collision cell. The sample processing procedure included liquid-liquid extraction, derivatization with dansyl chloride and a final cation-exchange extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (20-300 ng/mL), accurate and precise for the differential quantification of glutamic acid in the diffusion medium of retina cells. This is the first report of using chemical derivatization to enhance MS/MS detection of the glutamic acid released in the diffusion medium of wild-type and mutant retina cells, under ischemic conditions.