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Milano, Italy

Righetti P.G.,Miles Gloriosus Academy | Sebastiano R.,Polytechnic of Milan | Citterio A.,Polytechnic of Milan
Proteomics | Year: 2013

CZE and CIEF of proteins have preceded, and accompanied, the birth of proteomics. Although they might not be fully exploited in massive proteomic analyses (especially those projects aiming at a deep discovery of possibly the entire proteome of a cell or subcellular organelles or biological fluids), it still has interesting features and advantages, especially with samples of limited heterogeneity and in the field of purity checking for recombinant DNA proteins meant for human consumption. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to analysis of proteins and peptides, while commenting on the constraints and caveats of the technique. The tutorial ends with an outlook on the future, which might be dominated by microchip electrophoresis, especially for ultrafast analyses of protein samples in a sieving mode, in presence of either sieving liquid polymers or firm gels polymerized within the microchannels. To this purpose, commercial instrumentation is already available on the market. This tutorial is part of the International Proteomics Tutorial Programme (IPTP 13). © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Esteve C.,University of Alcala | D'Amato A.,Polytechnic of Milan | Marina M.L.,University of Alcala | Garcia M.C.,University of Alcala | Righetti P.G.,Miles Gloriosus Academy
Electrophoresis | Year: 2013

Musa ssp. is among the world's leading fruit crops. Although a strong interest on banana biochemistry exists in the scientific community, focused on metabolite composition, proteins have been scarcely investigated even if they play an important role in food allergy and stability, are a source of biologically active peptides, and can provide information about nutritional aspects of this fruit. In this work we have employed the combinatorial peptide ligand libraries after different types of protein extractions, for searching the very low-abundance proteins in banana. The use of advanced MS techniques and Musa ssp. mRNAs database in combination with the Uniprot_viridiplantae database allowed us to identify 1131 proteins. Among this huge amount of proteins we found several already known allergens such as Mus a 1, pectinesterase, superoxide dismutase, and potentially new allergens. Additionally several enzymes involved in degradation of starch granules and strictly correlated to ripening stage were identified. This is the first in-depth exploration of the banana fruit proteome and one of the largest descriptions of the proteome of any vegetable system. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Boschetti E.,EB Consult | Righetti P.G.,Miles Gloriosus Academy
Electrophoresis | Year: 2012

Food science is a complex domain where modern analytical technologies interact with each other. Named as "Foodomics," this domain assembles a variety of investigational fields such as genomics, transcriptomics, proteomics, peptidomics, and metabolomics. All these technologies are at the service of several goals that are essentially dictated by fundamental understanding, safety, and consistency. In this report, open questions are summarized around proteins and selected technologies recommended for a proper analytical determination not only of composition, but also considering the low-abundance expressed species difficult to detect for their presence, their function, and their adverse effects such as allergic action. In particular, this review covers the proteome of foodstuff and beverages, as detected via combinatorial peptide ligand libraries, such as: bovine and donkey's milk, egg white, and yolk, white and red wine, beer, and a number of nonalcoholic beverages, such as almond's milk and orgeat syrup, Cola, ginger ale, and vinegar. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Righetti P.G.,Miles Gloriosus Academy | Boschetti E.,EB Consult | Candiano G.,Dell
Journal of Proteomics | Year: 2012

The present review highlights recent progresses in the technique of combinatorial peptide ligand libraries (CPPL), a methodology that has much to offer for the detection of low- to very-low abundance proteins (nanograms/mL scale and below) in any proteome. In particular, advances in exploration of the urinary, plasma and tissue proteomes are discussed and evaluated. It is shown that when treating biological fluids, such as plasma, with CPLLs, the detection sensitivity, which in the control only reaches 10. ng/mL, can be enhanced to as high as 10. pg/mL, with an increment of sensitivity of three orders of magnitude. The possibility of using CPLLs as a two-dimensional pre-fractionation of any proteome is also evaluated: on the charge axis, CPLL capture can be implemented at no less than three different pH values (4.0, 7.2 and 9.3), thus permitting a capture of proteinaceous analytes bearing a net positive or net negative charge, respectively. When capture is performed in the absence of salts or at high levels of salts (of the Hofmeister series), one can favor the capture of hydrophilic vs. hydrophobic proteins, respectively. This would thus be a genuine 2D protocol, working on orthogonal separation principles (charge vs. hydrophobicity). As the horizon of CPLLs is expanding and its use is exponentially growing, we expect major breakthroughs in, e.g., biomarker discovery, a field that has suffered a decade of failures. © 2012 Elsevier B.V. Source

Righetti P.G.,Miles Gloriosus Academy
Electrophoresis | Year: 2013

This review, compiled for celebrating the decennial of the series of issues of Electrophoresis on bioanalysis, does not cover much heri (yesterday in Latin), since this was extensively described by my previous review Electrophoresis 2004, 25, 2111-2127, nor, for that matter cras (tomorrow in Latin) since making predictions on future scenarios is decidedly a nonscientific endeavor, but is concentrated onto hodie (today in Latin). Recent progress in the following fields is covered: SDS electrophoresis; IEF and the unraveling of the chemistry and composition of soluble carrier ampholytes; 2D maps; CE with microchip technology and some coatings of unique performance; chemistry and function of combinatorial peptide ligand libraries. This latter technique is becoming fundamental in the detection and identification of low-abundance proteins in proteome analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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