Isolation and identification of phosphorylated lysine peptides by retention time difference combining dimethyl labeling strategy

Protein phosphorylation plays essential roles in various biological procedures. Despite the well-established enrichment strategies for O-phosphoproteomics, the intrinsic acid lability of N–P phosphoramidate bond(phosphorylation of histidine, arginine and lysine) has impaired the progress of N-phosphoproteomics. Herein, we reported a retention time difference combining dimethyl labeling(ReDD) strategy for the isolation and identification of phosphorylated lysine(pLys) peptides. By such a method, pLys peptide could be isolated under 100000-fold interference of non-phosphorylated peptides. Furthermore, ReDD strategy was applied to map pLys sites from E. coli samples, leading to the identification of 11 pLys sites, among which K26p that originating from autonomous glycyl radical cofactor was validated both in mass spectrometry and HPLC co-elution experiments. Furthermore, 112 pLys sites from 100 proteins were identified in HeLa cells. All these results demonstrate that ReDD could provide a first glimpse into Lys phosphorylation, and could be an important step toward the global perspective on protein phosphorylation.     

Examination of intermediates in globular protein unfolding by the tritium labeling method

Unfolding of polypeptide chain of ribonuclease A in urea and guanidinium chloride (GuCl) solutions under equilibrium conditions involves formation of intermediates whose properties (compactness and preservation of the most of the native hydrophobic core, secondary structures, and native-like folding of the polypeptide chain) correspond to the basic characteristics of the “molten globule” state. Intermediates are “damp” molten globules (with water molecules inside the globule). The examinations performed revealed pronounced distinctions in the properties of the intermediates, above all, in their compactness degrees.     

A method for tentative identification of unknown gas chromatographic peaks by retention index

A method for gaining structural information from gas-liquid chromatographic or radiochromatographic data, using the structure-retention index relationship (SRIR), is reported. The method can be applied to predict the retention indexes of a wide range of apolar and polar compounds and the silylated derivatives of polar compounds.     

Facile and efficient postsynthetic tritium labeling method catalyzed by Pd/C in HTO

[reaction: see text] We have developed a facile and efficient tritium labeling method using a Pd/C-HTO-H2 system. This method can provide multitritium-labeled compounds in highly diluted HTO under T2 gas-free conditions, and is environmentally benign since purification by silica gel column chromatography is not necessary, which causes a large quantity of radioactive waste such as silica gel and eluent.     

Volume reduction and tritium retention factor in electrolytic enrichment of water

Volume reduction(N), tritium retention factor (R), tritium concentration factor(Z) and apparent separation factor(beta) were measured on the large and small electrolytic cell systems. The relative variation of R was smaller than that of Z. So, it is recommended to use R in calculation of tritium concentrations in water samples. Furthermore, it was empirically revealed that R can be obtained only from N if a reliable beta-value is previously known. Therefore, it is possible to obtain R without electrolysis of the tritium standard solution. Taking into account the above facts, the so-called non-spike analysis of tritium, in which electrolytic enrichment and liquid scintillation counting are combined, becomes practicable.     

Use of equivalent isothermal retention index for identification in gas chromatography with linear temperature programming

Conclusions The equivalent isothermal index which the authors proposed earlier enables substances to be identified reliably under conditions of gas Chromatographic analysis with linear temperature programming.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 3, pp. 679–680, March, 1970.     

A new technique for tritium labeling of complex technical mixture of PCB congeners

The method of labeling by thermally activated tritium of technical mixture of PCB congeners (Sovol) has been developed. Influence of labeling procedure on mixture of PCB congeners was investigated and optimum conditions of producing of tritium labeled complex mixture of PCB congeners were determined. The system purification of tritium labeled Sovol by thin layer chromatography was developed. Purified tritium labeled Sovol was analyzed by gas chromatography. The developed technique can be used for producing of tritium labeled similar complex mixtures of congeners of other organic compounds.     

Elemental labeling for the identification of proteinaceous-binding media in art works by ICP-MS

In the history of art, artists have used many different organic compounds to dissolve pigments and apply them onto a support to obtain a paint layer. Proteins were used with success from the Middle Ages up to the Renaissance, and the traditional protein sources were animal parts (skins, tendons and bones) or milk and eggs. Moreover, some of these materials are commonly used as adhesive. In this paper, the first application of the metallomic analytical technique to the identification of proteins in artworks is reported. Samples were derivatized with DTPA/Eu and the derivatization procedure was evaluated by matrix‐assisted laser desorption/ionization time‐of‐flight before high performance liquid chromatography inductively coupled plasma MS analysis. This study has been carried out on laboratory models prepared in‐house for method development, resulting in the correct identification of the different classes of proteinaceous binders typically used. In addition, some unknown paint layer samples have been analyzed demonstrating that the method is applicable to very small sample amounts (0.6 mg), which are compatible with the amount normally available for this kind of analysis. The results obtained demonstrate the effectiveness of the method, suggesting the potential future use as novel diagnostic tool in the scientific study of artworks. Copyright © 2011 John Wiley & Sons, Ltd.     

Gas-chromatographic identification of chloro- and bromo-substituted anilines by their retention indices

Gas chromatography retention indices were experimentally determined for 27 chloro- and bromo-substituted anilines on the standard non-polar polydimethylsiloxane stationary phase; a procedure for the estimation of retention indices for any other species of the studied class was proposed. The computational algorithm is based on a modified additive scheme equivalent to the assembly of the target structure from simpler molecules characterized by retention indices, using their addition and subtraction.     

Gas-chromatographic identification of products formed in iodination of methyl phenols by retention indices

Iodination reaction followed by conversion of iodine-substituted methylphenols to the corresponding trifl uoroacetates was suggested for improving the sensitivity of the gas-chromatographic determination of phenol and its methyl-substituted derivatives (al isomers of mono- and diethylphenols, 2,3,5-, 2,3,6-, and 3,4,5-trimethylphenols) in aqueous media. Acylation products of iodo methylphenols (104 compounds) were identified by linear-logarithmic retention indices on a standard nonpolar polydimethylsiloxane stationary phase, and the pattern of their variation with the number and nature of substituents were characterized. A procedure for identification of methyl-substituted phenols in water in their gas-chromatographic determination with an electron-capture detector was developed.     

The absolute retention index in chromatography. Part 1

A definition for an absolute retention index is given. This index is independent of whether the process is isothermal or temperature programmed. The relation between this index and Kovats's index is discussed. General equations are derived for the retention times of homologous series, from which the position of the air peak or missing members can be determined. Methods used in the literature for air peak determination are improved and extended to other cases of interest. The retention index in temperature programmed chromatography is reexamined in the light of recent publications on temperature programming.     

Glycan labeling strategies and their use in identification and quantification

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.