High-resolution separation of peptides by sodium dodecyl sulfate-polyacrylamide gel "focusing"

As a follow-up of our previous report (Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a refinement of the method for separation of peptides in the small to medium M(r) range (0.5-10 kDa) is here reported, based on a shallow gradient of immobilized positive charges (0-10 mM) onto a minimally sieving polyacrylamide gel matrix (4%T, 2.5%C). Unlike conventional SDS-PAGE, which rarely can achieve the separations of polypeptide chains below a critical value of 10 kDa, the present method can be fine-tuned to perform such separations even down to a size of only 500 Da. In the case of larger fragments, the major peptide zones are shown, under microscope observation, to be composed by envelopes of bands as narrow as 20-100 microm, spaced at regular intervals of 100-150 microm. It is hypothesized that such larger peptides could form complexes with rather small SDS micelles and that such peptide-SDS complexes could differ in charge by just a single negative charge.     

Gel-free IEF in a membrane-sealed multicompartment cell for proteome prefractionation

A minidevice for performing gel-free proteome prefractionation via conventional IEF in soluble carrier ampholyte buffers is reported here. It consists of a compact block of polyoxymethylene in which eight samples and two electrode chambers are machined. Each of the eight sample chambers can be filled with up to 120 microL of sample and has the following size: 7 mm width, 3 mm depth and 10 mm height. The anodic and cathodic compartments have the same width and height as the sample chambers, but with a depth of 6 mm, thus accepting up to 250 microL of electrodic solutions. Focusing is in general accomplished in 2 h with a voltage gradient of up to 1000 V (7 cm electrode distance). Easy fractionation and collection of the content of the eight chambers is achieved by simply pressing a rubber diaphragm against the edges of the thin walls separating each well, this automatically breaking liquid continuity. The performance of this device has been tested by subfractionating total cell lysates of a human cancer cell line (U2Os) and of Escherichia coli bacterial cells, and by analysing the content of each chamber by mono-dimensional SDS-PAGE and 2-D maps.     

Romancing the "hidden proteome", Anno Domini two zero zero seven

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides, for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins constituting the vast majority of species in any proteome, be it a cell or tissue lysate or a biological fluid, are here reviewed. Mechanisms of adsorption are evaluated, as well as different protocols for en bloc or sequential elution of the captured polypeptides. Examples are given of capture of proteins from serum, human platelet extracts, bacterial extract and egg white. The increment in detection of low-abundance species appears to be of at least four-fold as compared with untreated samples. One particular aspect of this capture is the adsorption of a high proportion of small peptides (in the Mr 600-8000 Da range) that are normally lost upon electrophoretic two-dimensional mapping. Such a peptide population, in human sera, may be of particular importance since it may contain protein cleavage products of diagnostic value.     

Enzyme-Mediated Preparation of (+)- and (–)-cis-α-Irone and (+)- and (–)-trans-α-Irone

The preparation of (−)- and (+)-trans-α-irone ( 1a and 1b , resp.) and of (+)- and (−)-cis-α-irone ( 1c and 1d , resp.) from commercially available Irone alpha ^® is reported. The relevant step in the synthetic sequence is the initial chromatographic separation of crystalline (±)-4,5-epoxy-4,5-dihydro-cis-α-irone ((±)- 5 ) from oily (±)-4,5-epoxy-4,5-dihydro-trans-α-irone ((±)- 4 ). The latter was subsequently converted, after NaBH₄ reduction, into the crystalline 3,5-dinitrobenzoate ester (±)- 8 , thus allowing a complete separation of the two corresponding diastereoisomeric alcohol derivatives. Suitable enantiomerically pure precursors of the desired products 1a – d were obtained by kinetic resolution of the racemic allylic alcohols derived from (±)- 5 and (±)- 8 , mediated by lipase PS (Amano). The last steps consisted of MnO₂ oxidation and removal of the epoxy moiety with Me₃SiCl/NaI in MeCN. External panel olfactory evaluation showed that (−)-cis-α-irone ( 1d ) has the finest and most distinct `orris butter' character.     

Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis

Several methods have been described in the literature for removal of DNA from protein samples prior to proteome analysis. They in general involve protein precipitation techniques. In other protocols, DNAse treatment is suggested prior to precipitation of proteins in excess acetone. All these methods have been evaluated and found to perform poorly in DNA removal, as illustrated by two-dimensional (2D) maps where horizontal and vertical sample streaking are still substantial. Such removal is in general necessary in tissue lysates and especially when analysing sub-cellular organelles, such as nuclei, where the high DNA levels strongly interfere with proteome analysis. Another method is proposed here for efficient DNA removal: two-phase extraction of DNA in chloroform/phenol/isoamyl alcohol, a procedure commonly used to rid DNA samples of protein contaminants, but rarely applied to protein preparation. This extraction is not very efficient if performed at slightly acidic to neutral pH values, but it performs extremely well at pH values of 9.5 or higher. The 2D maps thus obtained of Escherichia coli lysates as well as extracts from purified nuclei of eukaryotic cells are not only devoid of any vertical or horizontal streaking, but exhibit many more spots, especially in the alkaline region of the 2D gels, suggesting that these basic proteins were in general lost to proteome analysis due to co-precipitation in tenacious protein–DNA complexes. It is hypothesized that the alkaline pH values adopted in the two-phase extraction help to fully disrupt any residual DNA–protein complexes, due to strong Coulombic repulsion.     

Dynamic switching between binding sites in the complexation of macrocyclic ‘push-pull’ chromophores to lanthanides

The introduction of a 1,3-diacetylpyridine moiety, as an additional binding niche in a macrocyclic receptor ligand containing a conjugated, push-pull malonate functionality, leads to, in addition to the 1:1 binding stoichiometry, the equilibrium formation of multiple complexes, specifically 1:2, 2:1, and 3:1 ligand:metal complexes with lanthanide trications. Various binding modes elicit distinctive responses in the visible region, which enhance the potential recognition of lanthanide ions. This paper also highlights the usefulness of a full factor analysis in the elucidation of complex binding phenomena.     

Fundamental properties of isoelectric buffers for capillary zone electrophoresis

Different isoelectric buffers are analysed theoretically, taking into account a fundamental parameter, i.e., the ratio between intrinsic buffering power and conductivity (R=β/λ). For a model ampholyte, the above parameter is analysed both as a function of the pI and the pK_b–pK_a values. For natural pH gradients, the variation of R, connected with approaching the isoelectric point, is evaluated. A case of oligo-protic ampholytes is also considered.     

Immobilized pH 2.5-11 gradients for two-dimensional electrophoresis.

Extremely wide immobilized pH gradients, pH 2.5-11, for isoelectric separation of complex protein mixtures are described. These pH gradients are theoretically and practically the maximum that can be achieved at present with the available acrylamido buffers and titrants. Conditions are described for reducing conductivity and electroendosmosis in extreme pH ranges. Furthermore, new conditions are described for the separation of proteins in the second dimension. Using this protocol, nearly all the possible cellular products can be separated in one single two-dimensional map.