Plasma proteomics for biomarker discovery: A study in blue
Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Milano, Italy. Electrophoresis
(Impact Factor: 3.03).
12/2011; 32(24):3638-44. DOI: 10.1002/elps.201100307
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.
Available from: Ann-Kristin Henning
- "After incubation with a settled bed volume of 100 lL Blue Sepharose 6 Fast Flow beads for 1 h at 20 °C with mild agitation, the beads were sedimented by centrifugation at 2000 g for 2 min and the supernatants were transferred to new tubes. Bound proteins were recovered after three washes with 1.0 mL incubation buffer by boiling with 100 lL 4% SDS and 25 mM DTT (Di Girolamo and Righetti, 2011). Albumin depletion by extraction with CB-Sepharose was tested with human, bovine and porcine samples under various conditions, including variation of the pH (see Appendix A: Supplementary Fig. 2), salinity and the polarity of the buffer (data not shown). "
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ABSTRACT: In this study, the bovine plasma proteome was analysed using a three step protocol: (1) plasma was treated with a combinatorial peptide ligand library (CPLL) to assimilate the differences in concentrations of different proteins in raw plasma; (2) CPLL-treated material was fractionated by three standard electrophoretic separation techniques, and (3) samples were analysed by nano-liquid chromatography (nLC) matrix-assisted laser desorption/ionisation (MALDI) time-of-flight tandem (TOF/TOF) mass spectrometry. The efficiencies of three fractionation protocols for plasma proteome analysis were compared. After size fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), resolution of proteins was better and yields of identified proteins were higher than after charge-based fractionation by preparative gel-free isoelectric focussing. For proteins with isoelectric points >6 and molecular weights ⩾63kDa, the best results were obtained with a 'shotgun' approach, in which the CPLL-treated plasma was digested and the peptides, rather than the proteins, were fractionated by gel-free isoelectric focussing. However, the three fractionation techniques were largely complementary, since only about one-third of the proteome was identified by each approach.
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ABSTRACT: Low-Abundance Proteome Discovery addresses the most critical challenge in biomarker discovery and progress: the identification of low-abundance proteins. The book describes an original strategy developed by the authors that permits the detection of protein species typically found in very low abundance and that may yield valuable clues to future discoveries. Known as combinatorial peptide ligand libraries, these new methodologies are one of the hottest topics related to the study of proteomics and have applications in medical diagnostics, food quality, and plant analysis. The book is written for university and industry scientists starting proteomic studies of complex matrices (e.g., biological fluids, biopsies, recalcitrant plant tissues, foodstuff, and beverage analysis), researchers doing wet chemistry, and graduate-level students in the areas of analytical and biochemistry, biology, and genetics. - Covers methodologies for enhancing the visibility of low-abundance proteins which, until now, has been the biggest challenge in biomarker progress - Includes detailed protocols that address real-life needs in laboratory practice - Addresses all applications, including human disease, food and beverage safety, and the discovery of new proteins/peptides of importance in nutraceutics - Compiles the research and analytic protocols of the two scientists who are credited with the discovery of these landmark methodologies, also known as combinatorial peptide ligand libraries, for the identification of low-abundance proteins.
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ABSTRACT: Immunization with complex mixtures, like the human plasma resulted in the generation of cloned mAb libraries (PlasmaScan™ and QuantiPlasma™ libraries, with >1000 individual mAbs) reacting with a non-redundant set of antigenic epitopes. Monoclonal antibody proteomics refers to quasi hypothesis free profiling of plasma samples with nascent or cloned mAb libraries for the discovery of disease specific biomarkers. Once mAbs with biomarker potential have been identified, the next task is the determination of cognate antigens recognized by the respective mAbs. To determine the cognate protein antigen corresponding to each individual mAbs in the cloned mAb libraries, we have separated human plasma by consecutive steps of desalting and various chromatography procedures. The process resulted in 783 fractions, which we termed "Analyte Library". The Analyte Library represents the human plasma proteome in relatively low protein complexity fractions. Here, to determine the utility of the Analyte Library, we selected ten plasma proteins and checked for their presence in the fractions. Among the ten cases, the distribution of four selected plasma proteins matched expectations, as these proteins were present only in a few fractions corresponding to their physical, chemical and biochemical properties. However, in six cases, we observed 'smear'-like distribution or complete absence of the proteins, suggesting that protein-protein interactions or protein variants may alter the observed plasma distribution profiles. Nevertheless, we conclude that the Analyte Library is an efficient, high throughput tool to complement the mAb biomarker discovery process with cognate protein antigen identification for each mAbs. This article is protected by copyright. All rights reserved.
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