Pier Giorgio Righetti

Publications (246)889.86 Total impact

• Source

  Available from: Massimo Donadelli

  Dataset: Proteomic analysis of pancreatic endocrine tumor cell lines treated with the histone deacetylase inhibitor trichostatin A

  Daniela Cecconi, Massimo Donadelli, Sara Rinalducci, Lello Zolla, Maria Teresa Scupoli, Aldo Scarpa, Marta Palmieri, Pier Giorgio Righetti
• Article: Capillary electrophoresis and isoelectric focusing in peptide and protein analysis: a tutorial(*).

  Pier Giorgio Righetti, Roberto Sebastiano, Attilio Citterio
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  ABSTRACT: Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (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 rDNA (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 ultra-fast analyses of protein samples in a sieving mode, in presence of either sieving liquid polymers or firm gels polymerized within the micro-channels. To this purpose, commercial instrumentation is already available on the market. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 13).
  Proteomics 11/2012; · 4.43 Impact Factor
• Article: In-depth proteomic analysis of banana (Musa spp.) fruit with combinatorial peptide ligand libraries.

  Clara Esteve, Alfonsina D'Amato, María Luisa Marina, María Concepción García, Pier Giorgio Righetti
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  ABSTRACT: Musa ssp. is among the world 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 mass spectrometry techniques and musa 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 musa 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.
  Electrophoresis 10/2012; · 3.30 Impact Factor
• Article: Artichoke and Cynar liqueur: Two (not quite) entangled proteomes.

  Vicente Saez, Elisa Fasoli, Alfonsina D'Amato, Ernesto Simó-Alfonso, Pier Giorgio Righetti
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  ABSTRACT: Combinatorial peptide ligand libraries (CPLLs) have been adopted to investigate the proteome of artichoke extracts, of a home-made alcoholic infusion and of the Italian Cynar liqueur. The aim of study was not only to perform the deepest investigation so far of the artichoke proteome but also to assess the genuineness of the commercial aperitif via a three-pronged attack. First, different extraction techniques have been used for the characterization of the artichoke's proteome, secondly a home-made infusion has been analyzed and finally the proteome of the commercial drink was checked. The artichoke proteome has been evaluated via prior capture with CPLLs at four different pH (2.2, 4.0, 7.2 and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 876 unique gene products in the artichoke extracts, 18 in the home-made infusion and no proteins at all in the Italian Cynar liqueur, casting severe doubts on the procedure stated by the manufacturer (that should be made by an infusion of artichoke leaves plus thirteen different herbs). This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.
  Biochimica et Biophysica Acta 09/2012; · 4.66 Impact Factor
• Article: Identification of avocado (Persea americana) pulp proteins by nano-LC-MS/MS via combinatorial peptide ligand libraries.

  Clara Esteve, Alfonsina D'Amato, María Luisa Marina, María Concepción García, Pier Giorgio Righetti
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  ABSTRACT: Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse-phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low-abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone reductase like protein.
  Electrophoresis 09/2012; 33(18):2799-805. · 3.30 Impact Factor
• Article: Breakfast at Tiffany's? Only with a low-abundance proteomic signature!

  Egisto Boschetti, Pier Giorgio Righetti
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  ABSTRACT: 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.
  Electrophoresis 08/2012; 33(15):2228-39. · 3.30 Impact Factor
• Article: Allergomic study of cypress pollen via combinatorial peptide ligand libraries.

  Youcef Shahali, Jean-Pierre Sutra, Elisa Fasoli, Alfonsina D'Amato, Pier Giorgio Righetti, Norihiro Futamura, Egisto Boschetti, Hélène Sénéchal, Pascal Poncet
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  ABSTRACT: Although Cupressus sempervirens (Cups) pollen represents one of the main aeroallergens in southern Europe, only two Cups allergens have yet been identified and reported: Cup s 1 and Cup s 3. The aim of this study was to identify allergens in cypress pollen using an immuno-proteomic approach. A sequential pollen protein extraction was developed and supplemented by a combinatorial peptide ligand library (CPLL) treatment to select low-abundance species. Control extracts and CPLL eluates have then been resolved by 1-DE and 2-DE gel electrophoresis, blotted and confronted with sera from cypress allergic patients. Extracted proteins including IgE-binding components were identified using nanoLC-MS/MS analysis. A total of 108 unique gene products were identified analyzing the eluates and control loaded onto 1-DE SDS-PAGE. Forty proteins were identified in control samples and 68 supplementary species upon CPLL treatment. Out of the 12 IgE-binding proteins characterized in 2-DE gels, 9 were already reported as allergens in various sources including the two major known allergens of Cupressaceae (groups 1 and 2). Three IgE-binding proteins, not previously reported as allergens, are newly described. The improvement in protein extraction combined with the enrichment of low-abundance species allowed us to extend the repertoire of potential cypress pollen allergens.
  Journal of proteomics 07/2012; · 5.07 Impact Factor
• Article: Mark Twain: how to fathom the depth of your pet proteome.

  Pier Giorgio Righetti, Egisto Boschetti, Giovanni Candiano
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  ABSTRACT: 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.
  Journal of proteomics 06/2012; 75(15):4783-91. · 5.07 Impact Factor
• Article: Assessment of the floral origin of honey via proteomic tools.

  Francesco Di Girolamo, Alfonsina D'Amato, Pier Giorgio Righetti
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  ABSTRACT: The honey from chestnut, acacia, sunflower, eucalyptus and orange was analysed for its proteome content, in order to see if any plant proteins present would allow the proteo-typing of these different varieties. Since the total protein content turned out to be minute, 200g of each honey type were diluted to 1L and then added with ProteoMiner to enhance the visibility of the proteinaceous material. All bands visible in the SDS-PAGE profile of each type of honey were eluted, digested and identified by mass spectrometry in a LTQ-XL instrument. It turned out that all proteins identified (except one, the enzyme glyceraldehyde-3-phosphate dehydrogenase from Mesembryanthemum crystallinum) were not of plant origin but belonged to the Apis mellifera proteome. Among the total proteins identified (eight, but only seven as basic constituents of all types of honey) five belonged to the family of major royal jelly proteins 1-5, and were also the most abundant ones in any type of honey, together with α-glucosidase and defensin-1. It thus appears that honey has a proteome resembling the royal jelly proteome (but with considerably fewer species), except that its protein concentration is lower by three to four orders of magnitude as compared to royal jelly. Attempts at identifying additional plant (pollen, nectar) proteins via peptidome analysis were unsuccessful.
  Journal of proteomics 04/2012; 75(12):3688-93. · 5.07 Impact Factor
• Article: Anyone for an aperitif? Yes, but only a Braulio DOC with its certified proteome.

  Elisa Fasoli, Alfonsina D'Amato, Attilio Citterio, Pier Giorgio Righetti
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  ABSTRACT: The trace proteome of a Braulio aperitif (a 21% alcohol beverage, named after a mountain in the Val di Stelvio, Italy) has been investigated via capture with combinatorial peptide ligand libraries (CPLL, ProteoMiner). This aperitif is made with an infusion of 13 mountain herbs and berries, among which four are officially indicated in the label: Achillea moschata, juniper (Juniperus communis subsp. alpina) berries, absinthe (Artemisia absinthium) and gentian (Gentiana alpina) roots. Via capture with CPLLs at pH 7.0 and 2.2 we were able to identify 29 unique gene products, among which the PR5 (parasite resistance) allergen Jun r 3.2, a 25kDa species from Juniperus rigida. Due to the paucity of data on these alpine herbs, it was difficult to attribute these proteins to the specific plant extracts presumably present in this beverage; however most of the species identified indeed belong to alpine herbs and plants, living in a habitat between 1000 and 2000m of elevation. Most of them are enzymes, spanning a Mr range from 10 to 65kDa. It is hoped that such a proteomic signature should help tracking counterfeited products sold on the market.
  Journal of proteomics 04/2012; 75(11):3374-9. · 5.07 Impact Factor
• Article: The Silk Road, Marco Polo, a Bible and its proteome: a detective story.

  Lucia Toniolo, Alfonsina D'Amato, Riccardo Saccenti, Davide Gulotta, Pier Giorgio Righetti
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  ABSTRACT: Around the end of XIII century (at the time of young Marco Polo's first trip to China at the court of Khubilai Khan in Khan Baliq) a pocket Bible was delivered by a Franciscan friar to the Mogul Emperor, in the framework of the evangelization program of the Far East. Four centuries later, in 1685, this Bible was rediscovered by the Jesuit Philippe Couplet in the house of a rich Chinese in Nanchin and donated to Cosimo III, Grand Duke of Tuscany. This Bible was recently "unearthed" in the Biblioteca Medicea Laurenziana in Florence, wrapped up in a precious yellow silk cloth, in a rather ruined state. After two years of restoration, the Bible will return to China in 2012 for a celebration of its >700years of life and of its remarkable return trip on the Silk Road. On account of the thinness of the parchment (barely 80μm thickness, the size of each foil being 16.5×11cm) it was widely held that the pages were produced from foetal lambskins. On tiny fragments of the margins of a foil, after several unsuccessful attempts at digesting the vellum, we were able to obtain a tryptic peptide mixture, which, upon mass spectrometry analysis, yielded the identity of 8 unique proteins, belonging to the genus Bos taurus, thus confirming the origin of the vellum from calfskins rather than from foetal lambskins. Our results prove that it is possible to obtain reliable protein extraction and IDs from ancient parchment documents.
  Journal of proteomics 04/2012; 75(11):3365-73. · 5.07 Impact Factor
• Article: Exploration of the sea urchin coelomic fluid via combinatorial peptide ligand libraries.

  Elisa Fasoli, Alfonsina D'Amato, Pier Giorgio Righetti, Rainer Barbieri, Daniele Bellavia
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  ABSTRACT: The urchin Paracentrotus lividus has been characterized via previous capture and enhancement of low-abundance proteins with combinatorial peptide ligand libraries (CPLL, ProteoMiner). Whereas in the control only 26 unique gene products could be identified, 82 species could be detected after CPLL treatment. Due to the overwhelming presence of two major proteins-the toposome (a highly glycosylated, modified calcium-binding, iron-less transferrin) and the major yolk proteins, belonging to the class of cell adhesion proteins-which constituted about 70% of the proteome of this biological fluid and strongly interfered with the capture of the minority proteome, no additional proteins could be detected. Yet, at present, this constitutes the most thorough investigation of the proteome of this biological fluid.
  Biological Bulletin 04/2012; 222(2):93-104. · 1.70 Impact Factor
• Article: Ginger Rogers? No, Ginger Ale and its invisible proteome.

  Elisa Fasoli, Alfonsina D'Amato, Attilio Citterio, Pier Giorgio Righetti
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  ABSTRACT: The trace proteome of a Ginger drink, stated to be produced with a ginger root extract, has been investigated via capture with combinatorial peptide ligand libraries (ProteoMiner). Although in traces, we could confirm the presence of five grape proteins and one apple protein, but not even the faintest trace of any ginger root proteins. The first two findings are correct, as the producer stated that this beverage had been reinforced with 12% grape juice and 6% apple juice, but the absence of even traces of ginger proteins does not permit the classification of this beverage as a ginger extract on a proteomics scale. However, organoleptic tasting has confirmed the presence of a ginger extract, due to its piquant and tongue-biting taste. Nevertheless, any ginger root extract must be considered as a minor component as compared to the presence of grape and apple juice. At the light of these findings, it is hoped that the competent authorities will in the future make compulsory the proper labelling also of beverages so that all amounts of compounds utilized will be clearly stated in the label, including the presumptive main component.
  Journal of proteomics 03/2012; 75(6):1960-5. · 5.07 Impact Factor
• Article: Identification of olive (Olea europaea) seed and pulp proteins by nLC-MS/MS via combinatorial peptide ligand libraries.

  Clara Esteve, Alfonsina D'Amato, María Luisa Marina, María Concepción García, Attilio Citterio, Pier Giorgio Righetti
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  ABSTRACT: Different types of extraction protocols are described for identifying proteins in seed and pulp of olive (Olea europea), by employing both conventional extraction methods and capture with ProteoMiner as well as with in house-made combinatorial peptide ligand libraries (HM-CPLLs) at pH 7.4 and at pH 2.2. Thanks to the use of CPLLs, able to dramatically amplify the signal of low-abundance species, a quite large number of compounds has been indeed identified: 61 in the seed (vs. only four reported in current literature) and 231 in the pulp (vs. 56 described so far), the deepest investigation up to the present of the olive proteome. In the seed, it highlights the presence of seed storage proteins, oleosins and histones. In the pulp, the allergenic thaumatin-like protein (Ole e 13) was confirmed, among the other 231, as the most abundant protein in the olive pulp. The present research has also been undertaken with the aim of identifying proteins in olive oil and ascertaining the relative contribution of seed and pulp proteins in their presence, if any, in oils.
  Journal of proteomics 02/2012; 75(8):2396-403. · 5.07 Impact Factor
• Article: "Cheek-to-cheek" urinary proteome profiling via combinatorial peptide ligand libraries: A novel, unexpected elution system.

  Giovanni Candiano, Laura Santucci, Maurizio Bruschi, Andrea Petretto, Chiara D'Ambrosio, Andrea Scaloni, Pier Giorgio Righetti, Gian Marco Ghiggeri
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  ABSTRACT: A new method is here reported for facile elution of the human urinary proteome after being captured with combinatorial peptide ligand libraries (CPLL, ProteoMiner). It consists in challenging the beads with 100mM Tris, pH 7.4, or with 100mM Lys, pH 7.4 or even better with a mixture of Lys, Arg, Asp and Glu (150mM final concentration). These elutions permit recovery of species in a native form, for monitoring any biological activity of the eluted species, while avoiding the noxious presence of sodium dodecyl sulphate (SDS), reported as the best eluant so far from CPLL beads. SDS, albeit permitting quantitative recovery from the beads, has to be removed from the sample prior to mass spectrometry analysis. This unorthodox elution, which most likely will work only for urine samples, seems to be due to the fact that bile salts and urinary pigments are massively adsorbed by the beads, thus masking the hydrophobic binding sites of aromatic and non-aromatic amino acids. The binding thus occurs mostly via ionic and hydrogen bond interactions via the "Grand Catchers" Arg, Lys, His, which can then be easily challenged by positively charged species, such a Tris, free Lys and free Arg in the eluant as well as by negatively charged compounds, such as Glu and Asp. When eluting with the four-amino acid mix, at least 3300 spots can be visualized in a 2D map.
  Journal of proteomics 01/2012; 75(3):796-805. · 5.07 Impact Factor
• Article: Harry Belafonte and the secret proteome of coconut milk.

  Alfonsina D'Amato, Elisa Fasoli, Pier Giorgio Righetti
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  ABSTRACT: The proteome of coconut milk has been extensively mapped via capture at three pH values with combinatorial peptide ligand libraries (CPLL). A grand total of 307 unique gene products could be listed, 200 discovered via CPLL capture, 137 detected in the control, untreated material and 30 species in common between the two sets of data. This is by far the most extensive mapping of coconut milk, in which, up to the present, only a dozen proteins were known, those belonging to the high- to very-high abundance class. The database of coconut contains only 106 proteins: of those, only six are listed in our table. The vast majority of the classified proteins, thus, has been identified only by homologies with sequences deposited in the general viridiplantae database. This unique set of data could be the starting point for nutritionists and researchers involved in nutraceutics for enucleating some proteins responsible for some of the unique beneficial health effects attributed to coconut milk.
  Journal of proteomics 01/2012; 75(3):914-20. · 5.07 Impact Factor
• Article: Resurrexit, sicut dixit, alleluia. Snake venomics from a 26-year old polyacrylamide focusing gel.

  Pier Giorgio Righetti, Bruno Lomonte, Juan J Calvete
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  ABSTRACT: A 26-year-old dried polyacrylamide gel, cast in presence of an immobilized pH gradient and containing focused proteins from the venoms of a northern black-tailed rattlesnake (Crotalus molossus molossus), and of a western diamondback rattlesnake (Crotalus atrox) has been screened in order to see the feasibility of extracting the proteins, analyzing them by mass spectrometry (MS) and assessing their integrity. Nine gel bands were excised along the pH 3-10 gradient and the gel segments reswollen in warm acetonitrile. Upon digestion and MS analysis, all the bands could be identified and attributed to the respective venoms of the two rattlesnake species. Although a few peptides exhibited modified amino acids, the proteins were found to be well preserved even upon such a long storage at room temperature. The present data suggest the feasibility of identifying proteins from very old samples trapped in polyacrylamide gels, and analyzed in a pre-mass spectrometry era, thus of uncertain identity.
  Journal of proteomics 01/2012; 75(3):1074-8. · 5.07 Impact Factor
• Article: Polar electrophoresis: shape of two-dimensional maps is as important as size.

  Renato Millioni, Rita Polati, Michele Menini, Lucia Puricelli, Manuela Miuzzo, Paolo Tessari, Enrico Novelli, Pier Giorgio Righetti, Daniela Cecconi
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  ABSTRACT: The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of "isobaric" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.
  PLoS ONE 01/2012; 7(1):e30911. · 4.09 Impact Factor
• Article: "The quest for biomarkers": are we on the right technical track?

  Egisto Boschetti, Maxey C M Chung, Pier Giorgio Righetti
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  ABSTRACT: The discovery phase of biomarkers of diagnostic or therapeutic interest started a decade ago with the very rapid development of proteomic investigations. In spite of the development of innovative technologies and multiple approaches, the "harvest" is still modest. Various reasons justified the encountered difficulties and most of them have been circumvented by specific sample treatments or dedicated analytical approaches. Nevertheless, the situation of very modest biomarker discovery level did not change much. This review intends to specifically analyze the main approaches used for biomarker discovery phase and evaluate related advantages and disadvantages. Thus, preliminary sample treatments such as fractionation, depletion and reduction of dynamic concentration range will critically be discussed and then the main differential expression investigation methods analyzed. Combinations of technologies are also discussed along with possible proposals to federate associations of complementary technologies for better chances of success.
  PROTEOMICS - CLINICAL APPLICATIONS 12/2011; 6(1-2):22-41. · 1.81 Impact Factor
• Article: Plasma proteomics for biomarker discovery: a study in blue.

  Francesco Di Girolamo, Pier Giorgio Righetti
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  ABSTRACT: The performance of Cibacron Blue dye (HiTrapBlue or Affigel Blue) in depleting albumin from plasma, as a pre-treatment for biomarker searching in the low-abundance proteome, is here assessed. It is shown that (i) co-depletion of non-albumin species is an ever-present hazard; (ii) the only proper eluant able to release quantitatively the proteins bound to the dye is boiling 4% SDS-25 mM DTT, an ion shock (2 M NaCl) being quite ineffective in releasing the low-abundance species tightly bound to the dye moiety; (iii) the mechanism of dye-protein interaction, after an initial ion-ion docking, is a robust hydrophobic interaction, which progressively augments at lower and lower pH values; (iv) at pH 2.2 in the presence of 0.1% TFA, the blue resin behaves, for all practical purposes, just as a reverse-phase chromatography column, since all residual proteins present in plasma are completely harvested. However Cibacron Blue technology should not necessarily be discarded: As long as also the plasma fraction adsorbed is properly released and analyzed, together with the flow through, one should be able to perform a viable analysis of the low-abundance proteome.
  Electrophoresis 12/2011; 32(24):3638-44. · 3.30 Impact Factor