Protein Equalizer™ Technology : The quest for a “democratic proteome”

Abstract

No proteome can be considered "democratic", but rather "oligarchic", since a few proteins dominate the landscape and often obliterate the signal of the rare ones. This is the reason why most scientists lament that, in proteome analysis, the same set of abundant proteins is seen again and again. A host of pre-fractionation techniques have been described, but all of them, one way or another, are besieged by problems, in that they are based on a "depletion principle", i.e. getting rid of the unwanted species. Yet "democracy" calls not for killing the enemy, but for giving "equal rights" to all people. One way to achieve that would be the use of "Protein Equalizer Technology" for reducing protein concentration differences. This comprises a diverse library of combinatorial ligands coupled to spherical porous beads. When these beads come into contact with complex proteomes (e.g. human urine and serum, egg white, and any cell lysate, for that matter) of widely differing protein composition and relative abundances, they are able to "equalize" the protein population, by sharply reducing the concentration of the most abundant components, while simultaneously enhancing the concentration of the most dilute species. It is felt that this novel method could offer a strong step forward in bringing the "unseen proteome" (due to either low abundance and/or presence of interference) within the detection capabilities of current proteomics detection methods. Examples are given of equalization of human urine and serum samples, resulting in the discovery of a host of proteins never reported before. Additionally, these beads can be used to remove host cell proteins from purified recombinant proteins or protein purified from natural sources that are intended for human consumption. These proteins typically reach purities of the order of 98%: higher purities often become prohibitively expensive. Yet, if incubated with "equalizer beads", these last impurities can be effectively removed at a small cost and with minute losses of the main, valuable product.

Quantitative matrix-assisted laser desorption/ionization mass spectrometry

“…This can enhance coverage, but the reproducibility and throughput of these additional steps must be carefully evaluated. Several strategies have been reported [18][19][20], but these are yet to be comprehensively evaluated and other reproducible, high-throughput sample fractionation methods are certainly needed.…”

Exploring the venom proteome of the African puff adder, Bitis arietans, using a combinatorial peptide ligand library approach at different pHs

“…To minimize this problem and allow access to the ''deep proteome'', a number of multidimensional pre-fractionation and tools for immunodepleting the most abundant proteins have been described [2][3][4]. Further, the use of a combinatorial library of hexapeptide ligands (Equalizer beads or ProteoMiner™, Bio-Rad) has been shown to allow access to low-abundance proteins undetectable by classical analytical methods [5][6][7][8]. The use of large peptide libraries under overloading conditions to capture and "amplify" low abundance proteins from the whole proteome is based on a seminal paper by Thulasiraman et al [9] and has been applied by PG Righetti´s group for exploring the "hidden proteome" of a number of biological samples, including human urine [10] and serum [11], human platelets [12], human erythrocytes [13], chicken egg white [14] and yolk [15], plant tissues [16], and for detecting rare protein impurities [17,18].…”

Plant proteomics in India and Nepal: current status and challenges ahead

“…Another strategy is to use affinity approach to enrich rare or desired class of proteins. Recent development in this area is the use of combinatorial peptide ligand libraries (CPLL), which has been used to detect LAPs from a variety of biological samples (Righetti et al 2006(Righetti et al , 2011, including plants (Frohlich et al 2012) .…”