Improved procedure for protein binder analysis in mural painting by LC-ESI/Q-q-TOF mass spectrometry: detection of different milk species by casein proteotypic peptides

Diagnostic techniques applied to the field of cultural heritage represent a very important aspect of scientific investigation. Recently, proteomic approaches based on mass spectrometry coupled with traditional spectroscopic methods have been used for painting analysis, generating promising results for binder’s protein identification. In the present work, an improved procedure based on LC-ESI/Q-q-TOF tandem mass spectrometry for the identification of protein binders has been developed for the molecular characterization of samples from an early-twentieth-century mural painting from the St. Dimitar Cathedral in Vidin, Bulgaria. The proteomic investigation has led to the identification of both egg white and egg yolk proteins, according to traditional old recipes for tempera paintings. In addition, beyond the egg components, the presence of caseins was also revealed, thus suggesting the use of milk as binding medium, fixative or stabilising agent. Furthermore, for the first time, the capability to discriminate the milk origin on the basis of alpha casein proteotypic peptides is reported, that are diagnostic for a given species, thus opening interesting perspectives in art and archaeological fields.     

A rapid method for determining the casein content of milk

A method is presented for the rapid determination of the casein content of milk by Direct Injection Enthalpimetry (DIE).A significant heat pulse of precipitation of casein is produced by adjusting the pH of the solution to the pH of the isoelectric point of casein. The method is calibrated against a real sample whose casein content has been determined by a standard method incorporating the time consuming Kjeldahl method, but once calibrated the proposed method can be operated routinely by non-skilled personnel. The method is sensitive and gives results as acceptable as those obtained by standard methods. The method can be applied to most liquid samples without the need for prior preparation. the main advantages of the method are those of time and costs of analysis and the potential of the enthalpimetric method for automation.

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Zusammenfassung Es wird ein Verfahren zur Schnellbestimmung des Kaseingehaltes von Milch mittels Direct Injection Enthalpimetry (DIE) beschrieben.Durch das Einstellen des pH-Wertes der Lösung auf den pH-Wert des isoelektrischen Punktes von Kasein kann infolge des Ausfällens von Kasein ein eindeutiger Wärmeimpuls erzeugt werden. Das Verfahren wurde anhand einer wirklichen Probe geeicht, deren Kaseingehalt mittels dem zeitaufwändigen Kjeldahl-Verfahren bestimmt wurde. Einmal kalibriert kann das neue Verfahren routinehaft auch von Laien durchgeführt werden. Die Empfindlichkeit der Methode ist gut, die erhaltenen Ergebnisse können genauso akzeptiert werden, wie die in Standardverfahren erhaltenen Ergebnisse. Dieses Verfahren kann bei den meisten flüssigen Proben ohne jede vorherige Vorbereitung angewendet werden. Der Hauptvorteil besteht in der Ersparnis von Zeit und Analysenkosten und in der Anwendbarkeit des enthalpiemetrischen Verfahrens zur Automatisierung.

     

Validation of capillary electrophoresis in the analysis of ewe's milk casein

Recent investigations have shown that capillary electrophoresis (CE) can be an alternative to other techniques such as polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the qualitative analysis and separation of the different casein fractions in cow's and ewe's milk. However, past work has not yet clarified whether that method can achieve good quantifications. The present study has used a commercial whole ovine casein standard and a mixture of the standard and whole casein extracted from ewe's milk cheese to test the reliability of the technique. The results show that CE was able to quantify the ewe's milk caseins. The areas under four of the most representative peaks on the electrophoretogram for two alpha and two beta-caseins (designated alpha-casein1CE, alpha-casein2CE, beta-casein1CE, and beta-casein2CE in order of elution) were used to validate the method. In relation to linearity, coefficient of determination (r2) values greater than 99% were obtained for the regressions of each of the caseins. Moreover, each casein yielded response factors with a relative standard deviation (R.S.D.) of less than or equal to 5. The coefficients obtained in the day-to-day reproducibility analysis were higher than those for the same-day repeatability, but all the values were within acceptable limits. In the study of accuracy, the percentage recovery rates for the alpha-casein fractions were higher than those for the beta-casein fractions, hence quantification of the latter using this technique would appear to be more accurate under the conditions employed.     

Flocculation of diatomite by methylated milk casein in seawater

A new biodegradable flocculant was prepared from a common and inexpensive protein. Milk casein was methylated in a 0.05 M HCl methyl alcohol solution at room temperature. The methylated milk casein (MeCS), having a methylation degree of 81%, was applied to the separation or flocculation of diatomite in seawater (pH 8.1+/-0.1) at room temperature (18-23 degrees C). The flocculating ability of MeCS was evaluated by a sedimentation balance method (cumulative measurement method). The diatomite suspension was effectively flocculated by the addition of a small amount of MeCS (0.25 wt% of the diatomite weight). The results of sedimentation analysis showed that the size-frequency curve had a very sharp and high peak; thus the diatomite floc formed by MeCS had a rather uniform size. The settling velocity of diatomite floc at the appropriate MeCS dosages (0.25-2 wt% of diatomite) was about 3 x 10(-3) ms(-1).     

酪蛋白多肽的制备和色谱分离方法

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。     

The decontamination of industrial casein and milk powder by irradiation

The efficacy of gamma radiation decontamination of industrial casein, a milk protein utilized as a component of many food and non-food products has been studied. Low-fat milk powder was also included with a purpose to study the microflora survival in protein-rich materials. Microbial analysis of the samples prior to irradiation showed that the initial total viable count was higher than 6.0 log cfu g⁻¹ in both casein and milk powders. The contamination of casein with moulds and yeasts was found to be equal to 3.56 log cfu g⁻¹. The counts of coliforms have not exceeded the value of 2.48 log cfu g⁻¹. Radiation processing of casein and milk powder has substantially reduced the microbial population of all samples. The dose of 5 kGy was sufficient to reduce the total microflora and coliforms counts to the level permitted for food products. Survivals of microorganisms were analyzed by the generalized exponential equation, SF=exp[−D/D_o)^α]. Values of an exponent, α, standing for the dispersion parameter, were equal to 0.65 and 0.70 for microorganisms contaminating casein and milk powders, respectively. The numerical value of the dispersion parameter α<1 indicates the concave dependence of a logarithm of surviving fraction versus radiation dose. No difference in microflora survival in irradiated samples tested immediately and in samples stored for 1-month after irradiation has been noticed.     

Toward stereocontrolled, chemoenzymatic synthesis of unnatural peptides

An efficient, chemoenzymatic method for the multicomponent synthesis of unnatural tripeptides is presented. Development of a previously described procedure combines the diversity offered by multicomponent reactions with the selectivity of biocatalysts and allows the convenient introduction of varied amino acid moieties into the tripeptide scaffold, with control of the stereochemistry. Additionally, it allows the introduction of a methyl group to the amide nitrogen, leading to derivatives of N-methylated amino acids.     

Chemical speciation of radionuclides through the microbial process in soils

Environmental factors (nutrients and pH) influence the retention of Sr, Cs, and Ba as analogue of long-lived radionuclides by the microorganism. The effect of pH and concentration of organic nutrients are proved to be significant factors for retention of cations (Sr, Cs and Ba) by the microorganisms inoculated from surface soil. Microorganisms can affect the speciation of Sr, Cs, and Ba in the environment. Concentration of organic nutrients and pH are indicated to be the important environmental factors in the consideration of the migration of the radionuclides through microbial processes in the terrestrial environment.     

The potential of arsenic speciation in molluscs for environmental monitoring

This paper reports the assessment of total arsenic and six arsenic species (As(III), As(V), MMA, DMA, AsBet, AsCol) as contaminants of mussel samples collected around the island of Sardinia and in the Gulf of Venice. The samples were analysed using cation- and anion-exchange HPLC-HG-AFS for speciation and ICP-AES for the total As determination. To ensure the robustness of the routine analytical method, the technique was validated using a candidate reference material, BCR-710, and good agreement was obtained. It was recognised that higher total arsenic concentration in mussels does not necessarily result in higher toxicity of mussel samples.     

Toward plasmonic monitoring of surface effects on bacterial quorum-sensing

Biofilm formation is facilitated by cell–cell communication processes known as quorum sensing, which enable collective behavior and metabolic coordination. Surface topography and chemistry play a significant role in bacterial adhesion and biofilm formation, yet methods for monitoring quorum sensing in situ suffer limitations. Herein we suggest the use of surface-enhanced Raman scattering to study the effects of surface topography and chemistry on quorum sensing signals involved in biofilm growth.     

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels.Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels.By following the development of 1/l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of κ-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk.This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.     

NMR monitoring of chain-specific stability in heterotrimeric collagen peptides

NMR spectroscopy is used to investigate the heterotrimeric nature of a collagen model peptide. Two distinct peptide chains (A and B) were synthesized to model a site in heterotrimeric basement membrane type IV collagen. For NMR studies, four amino acids in the B chain were labeled with 15N/13C. Circular dichroism spectroscopy and differential scanning calorimetry thermal stability results on a solution with both A and B peptides (molar ratio 2A:1B) are consistent with the presence of one heterotrimeric triple-helical molecular species. Heteronuclear single quantum coherence experiments on homotrimers of the B peptide show trimer peaks which disappear at temperatures higher than 10 degrees C, while the 2A:1B mixture has trimer peaks with increased stability and altered chemical shifts. The reduction in the number of Leu trimer peaks from three to one and the increased stability of trimer resonances confirm the participation of B chains in an AAB heterotrimer molecule.     

Deviation from the mobile proton model in amino-modified peptides: implications for multiple reaction monitoring analysis of peptides

The study of peptide fragmentation is important to the understanding of chemical processes occurring in the gas phase and the more practical concern of peptide identification for proteomic analysis. Using the mobile proton model as a framework, we explore the effect of amino-group modifications on peptide fragmentation. Three aldehydes are used to transform the peptides' primary amino groups into either a dimethylamino or a heterocyclic structure (five- or six-membered). The observed fragmentation patterns deviate strongly from those observed for the analogous underivatised peptides. In particular, the a1 ion is the base peak in most tandem mass spectra of the derivatised peptides. The a1 ion intensity depends strongly on the N-terminal amino acid, with tyrosine and phenylalanine having the strongest enhancement. Despite the change in fragmentation patterns of the derivatised peptides, they still provide high-quality tandem mass spectra that, in many cases, are more amenable to database searching than the spectra of underivatised peptides. In addition, the reliable presence of the a1 ion facilitates rapid quantitative measurements using the multiple reaction monitoring approach.     

Non-chromatographic speciation analysis of arsenic and antimony in milk hydride generation atomic fluorescence spectrometry

A rapid, high sensitivity method has been developed for the determination of As(III), As(V), Sb(III) and Sb(V) in milk samples by using hydride generation atomic fluorescence spectrometry. The method is based on the leaching of As and Sb from milk through the sonication of samples with aqua regia followed by direct determination of the corresponding hydrides both before and after reduction with KI. It was confirmed by recovery experiments on spiked commercially available samples that neither the reduced nor the oxidized forms of the elements under study or mixtures of the two oxidation states were modified by the room temperature sample treatment with aqua regia. The methodologies developed provided 3σ limit of detection values of 8.1, 10.3, 5.4 and 7.7 ng l⁻¹ for As(III), As(V), Sb(III) and Sb(V) in the diluted samples. Average relative standard deviation values of 5.7, 5.5, 8.2 and 4.7% were found for determination of As(III), As(V), Sb(III) and Sb(V) in commercially available samples of different composition and origin containing from 3.5 to 13.6 ng g⁻¹ total As and from 4.9 to 11.8 ng g⁻¹ total Sb, it being confirmed that As(V) and Sb(V) are the main species present in the samples analyzed (62±5 and 73±5%, respectively). The time required to determine As and Sb species in milk involves 10 min sonication and 30 min prereduction but these steps can be carried out for several sample simultaneously. Additionally the fluorescence measurement step involves less than 20 min for three replicates of all the four measurements required. So, in less than 2 h it is possible to determine the content of As(III), As(V), Sb(III) and Sb(V) in four samples.     

Synthesis of cyclic peptides through hydroxyl side-chain anchoring

A general method was developed for the synthesis of serine or threonine containing cyclic peptides utilizing the β-hydroxyl side-chain of these residues as an anchor point to Wang resin. The peptide chain was assembled by conventional Fmoc/tBu solid-phase chemistry followed by palladium catalyzed exposure of the allyl protected C-terminus group and on-resin cyclization. The cyclic heptapeptide stylostatin 1 was prepared to demonstrate the utility of this technique.