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The cytoplasmic proteome of spinach leaves (Spinacia oleracea L) has been investigated with the help of commercially available (ProteoMiner) combinatorial peptide ligand libraries and with home-made ligand beads as prepared in our laboratory. The protein capture had been performed at three pH values (4.0, 7.0 and 9.3) and elution performed in 4% boiling SDS, 20mM DTT. The total number of unique gene products identified amounts to 322 proteins, of which 114 are in common with the control, untreated sample, 18 are present only in the control and 190 represent the new species detected with the help of all combined eluates and likely represent low-abundance species. This is the first in depth exploration of the spinach cytoplasmic proteome and might enable further studies on interaction, regulation and expression of proteins biological processes in combination or not with transcriptomics data., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Combinatorial peptide ligand libraries (CPLLs) have been adopted for harvesting and identifying traces of proteins present in red wines. Surprisingly, although it is stated that red wines are in general fined with egg albumin, for all Italian wines investigated (in the areas around Chiari and Verona as well as in the Chianti area) we find that the only fining agent used is bovine casein, just like in white wines. Although the typical levels of casein found range between 45 to 85μg/L, in one case as little as 3.8μg/L of casein could be detected, an extremely high level of sensitivity, close to our lower detection limit of 1μg/L reported for white wines. As a result of such treatments, very small amounts of residual proteins in red wines could be identified: essentially no residual grape proteins (except for thaumatin), but only traces of proteins from Saccharomyces cerevisiae and a few proteins from plant pathogens and fungi (e.g., Botryotinia fuckeliana, Sclerotinia sclerotiorum, Aspergillus aculeatus). Contrary to what has been found in white wines, the best capture efficiency with CPLLs has occurred at pH 7.2 and pH 9.3, with minimal capture at pH 3.3. The fact that such very low levels of fining agents can still be detected in treated red wines should be taken into consideration by winemakers in labelling their products and by EC rulers in issuing proper regulations., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The latest advances in combinatorial peptide ligand libraries, with their unique performance in discovering low-abundance species in proteomes, are reviewed here. Explanations of mechanism, potential applications, capture of proteomes at different pH values to enhance the total catch and quantitative elutions, such as boiling in the presence of 5% sodium dodecyl sulfate and 3% dithiothreitol are included. The reproducibility of protein capture among different experiments with the same batch of beads or with different batches is also reported to be very high, with coefficient of variations in the order of 10-20%. Miniaturized operations, consisting of capture with as little as 20 or even 5 microl of peptide beads are reported, thus demonstrating that the described technology could be exploited for routine biomarker discovery in a biomedical environment. Finally, it is shown that the signal of captured proteins is linear over approximately three orders of magnitude, ranging from nM to microM, thus ensuring that differential quantitative proteomics for biomarker discovery can be fully implemented, providing species do not saturate their ligands.

When capturing proteins via combinatorial peptide ligand libraries, a method well known for drastically reducing the concentration of high-abundance proteins and substantially magnifying the signal of low-abundance species, thus leading to the discovery of a large number of proteins previously undetected in proteomes, we had constantly noticed that there would be a loss of species initially present in the untreated sample, to the tune of 5%, up to 15% in some cases. Such losses are a nuisance and hamper to some extent the unique performance of the method. In order to verify if such losses could be reduced and also to understand some mechanisms of the capture process, we introduce here an important variant to the capture operation, up to the present carried out in physiological saline at pH 7.2. In this novel protocol, the binding step is conducted at three different pH values, namely the standard one at pH 7.2, plus two additional processes, at acidic (pH 4.0) and alkaline (pH 9.3) pH values. Indeed the capture process is more extensive, with a number of additional species captured at the two pH extremes in sera and other proteomes. Interestingly, at pH 4.0 newly detected proteins were mostly acidic, while at the alkaline pH additional protein species were more evenly distributed throughout the pI range towards the alkaline area. The role of pH in the complex mechanism of binding among the hexapeptide library and the various proteomes being analyzed is discussed and evaluated. Due to significant changes in protein patterns with pH, recommendations are thus made to increase the possibility to find additional gene products illustrated by two examples (snake venom and leaf protein extract). Keeping under control the environmental pH when facing reproducibility studies or for comparative proteomics profiling is also a general rule suggested by this study.

In contrast to the three to four sequential elution steps routinely adopted for recovering proteomes adsorbed onto combinatorial peptide ligand libraries, we report here two en bloc elution systems, which are able to achieve recoveries in the order of 95% in a single step. One consists of TUC (7 M urea, 2 M thiourea, 3% CHAPS) added with 40 mM formic acid, the other of TUC added with 25 mM cysteic acid (Cys-A). Although both systems are almost equally performing, the formic acid eluant has as a drawback, namely the potential to modify proteins by formylation of Ser and Thr residues. On the contrary, the Cys-A system is unreactive towards proteins. Additionally, Cys-A, due to its very low pI value (1.80) does not interfere with subsequent 2D map analyses since, during the first isoelectric focusing step (in general performed in immobilized pH gradients), it migrates to the anodic compartment and thus vacates the gel. Conversely, formic acid would mostly collect around pH 3 and acetic or citric acid, formerly used in the UCA (9 M urea, 50 mM citric acid) eluant, would condense around pH 4 in the focusing step, interfering thus with 2D map analyses. Elution by boiling SDS of the small amount of protein left over after three sequential elution steps in TUC and 25 mM Cys-A and analysis by nanoLC-MS/MS has demonstrated that these residual proteins are indeed a residue left over from proteins already eluted in TUC-Cys-A and not new species absent in the latter eluate.