Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches

The present review attempts to cover a number of methods that appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this excursus only to conventional two-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation (sodium dodecyl sulfate (SDS)-electrophoresis). The first and oldest method applied in 2D mapping is based on statistical analysis performed on sets of gels via powerful software packages, such as the Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately-running a number of replicas for control and treated samples, the merging and comparing between these two sets of data being accomplished via the softwares just mentioned. Recent developments permit analyses on a single gel containing mixed samples differentially labelled and resolved by either fluorescence or isotopic means. In one approach, a set of fluorophors, called Cy3 and Cy5, are selected for differentially tagging Lys residues, via a "minimal labelling" protocol. A variant of this, adopts a newer set of fluorophors, also of the Cy3 and Cy5 type, reacting on Cys residues, via a strategy of "saturation labelling". There are at present two methods for quantitative proteomics in a 2D gel format exploiting stable isotopes: one utilizes tagging Cys residues with [2H0]/[2H3]-acrylamide; the other one, also based on a Cys reactive compound, exploits [2H0]/[2H4] 2-vinylpyridine. The latter reagent achieves 100% efficiency coupled to 100% specificity. The advantages and limitations of the various protocols are discussed.     

Estimation of crude oil grade using time-resolved fluorescence spectra

Time-resolved fluorescence (TRF) spectra of six crude oils from the eastern province of Saudi Arabia were excited using a pulsed laser radiation at 250 nm and measured at specific time gates (TG) within the leading and trailing edges of the laser temporal pulse. The spectra showed the presence of a shoulder near 380 nm that systematically decreased in intensity from high-grade to low-grade crudes, and also from earlier to later TGs. The intensities of these shoulders are shown to be useful in estimating the grades of crude oils, particularly when the TRF spectra are measured at TGs within the leading edge of the laser temporal pulse. Contour diagrams depicting the shapes of the TRF spectra as function of TG (within the leading and trailing edges) are also presented to serve as true fingerprints of the crudes.     

Concept of multipole magnetic field rotation in ECRIS

The conventional type of magnetic well is formed by superposition of two types of magnetic field, axial bumpy field and radial multipole field. It is used to contain plasma that consists of neutrals, ions and electrons. These particles are in constant motion in the well and energetic electrons create plasma by violent collisions with neutrals and ions. The confined electrons are constantly heated by ECR technique in the presence of magnetic field. In this paper it has been shown theoretically that how the electron motion is influenced in terms of heating, containment and azimuthal uniformity of plasma, by the axial rotation of the multipole magnetic field [1,2]. Afterwards, the feasibility of achieving a rotating magnetic multipole field is discussed to some extent. And it is seen that it is not beyond the capability of the scientific community in the present scenario of the advanced technology. Presently, it can be achieved for lesser field and slightly larger size of the multipole electromagnet and can be used for improvement of the ECR ion source (ECRIS).     

Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.     

Investigation of some 2,3-didehydro amino acids by fast ion bombardment,chemical ionization and mass-selected collision-induced dissociation mass spectrometry

Four 2,3-didehydro amino acids were investigated by fast ion bombardment (FIB) and ammonia chemical ionization (NH₃-CI) mass Spectrometry. The protonated molecules formed by the two techniques were mass selected and their respective collision-induced dissociations (CIDs) in the translational energy range 5–200 eV (in the laboratory frame of reference) were investigated. No obvious quantitative differences between the FIB and NH₃-CI mass spectra of the (E)- and (Z)-isomers were observed. Different internal excitation of the protonated molecules formed by the two techniques and the role of CID data in clarifying this particular observation are discussed.     

Die Reaktion des Digalliumsubiodids R(I)Ga‐Ga(I)R [R = C(SiMe3)3] mit Lithium‐diphenylphosphanid – radikalische Öffnung der Ga‐Ga‐Bindung

The Reaction of the Digallium Subiodide R(I)Ga‐Ga(I)R [R = C(SiMe₃)₃] with Lithium Diphenylphosphanide – Radical Cleavage of the Ga‐Ga Bond The easily available organoelement digallium(II) subiodide R(I)Ga‐Ga(I)R ( 1 ) [R = C(SiMe₃)₃] reacted with two equivalents of lithium diphenylphosphanide in toluene by the replacement of both iodine atoms by two phosphanido groups. The product, [R(H)Ga‐P(C₆H₅)₂]₂ ( 2 ), contains a four‐membered Ga₂P₂ heterocycle without direct Ga‐Ga bonding interactions and the gallium atoms exclusively in an oxidation state of +III. They are attached to hydrogen atoms, which may result from a reaction of a reactive intermediate with the solvent.     

Alkylation power of free Immobiline chemicals towards proteins in isoelectric focusing and two-dimensional maps, as explored by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

A number of Immobilines, with pK 1.0-10.3, were incubated with two proteins, bovine alpha-lactalbumin (pI 4.80) and chicken egg lysozyme (pI 9.32), at pH approximately 9-10 and the resulting solutions were examined by matrix assisted laser desorption/ionization mass spectrometry. The reflectron mode of the same technique was also used to analyze a number of tryptic digests of some solutions. The extent and the number of detected alkylation sites associated with the acidic protein were found to be linearly proportional to the pK values of the investigated Immobilines, an effect which was less evident for the basic protein. The high resolution measurements of some tryptic digests indicate the cysteine residues as the likely sites of alkylation. The implications of the present data for isoelectric focusing separations on immobilized pH gradients and for two-dimensional maps are discussed.     

Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how?

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human alpha- and beta-globin chains, which possess only one (alpha-) or two (beta-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.