The erythrocyte cytoplasmic proteome is composed of 98% hemoglobin; the remaining 2% is largely unexplored. Here we used a combinatorial library of hexameric peptides as a capturing agent to lower the signal of hemoglobin and amplify the signal of low to very low abundance proteins in the cytoplasm of human red blood cells (RBCs). Two types of hexapeptide library beads have been adopted: amino-terminal hexapeptide beads and beads in which the peptides have been further derivatized by carboxylation. The amplification of the signal of low abundance and suppression of the signal of high abundance species were fully demonstrated by two-dimensional gel maps and nano-LC-MSMS analysis. The effect of this new methodology on quantitative information also was explored. Moreover using this approach on an LTQ-Orbitrap mass spectrometer, we could identify with high confidence as many as 1578 proteins in the cytoplasmic fraction of a highly purified preparation of RBCs, allowing a deep exploration of the classical RBC pathways as well as the identification of unexpected minor proteins. In addition, we were able to detect the presence of eight different hemoglobin chains including embryonic and newly discovered globin chains. Thus, this extensive study provides a huge data set of proteins that are present in the RBC cytoplasm that may help to better understand the biology of this simplified cell and may open the way to further studies on blood pathologies using targeted approaches. Molecular & Cellular Proteomics 7: 2254 -2269, 2008.Mature red blood cells (RBCs) 1 have a life span of approximately 120 days and are optimally adapted for oxygen and carbon dioxide as well as for proton transport. They consist of a plasma membrane that envelopes a viscous concentrated (33%) solution of proteins of which hemoglobin (Hb) constitutes approximately 98% of the global proteome. The absence of nucleus and the loss of cytoplasmic organelles allow the RBC passing through narrow capillaries, with a concomitant drastic shape change, to properly accomplish its most important biological tasks. A number of other vital functions present in RBCs are related to appropriate generation and expenditure of energy. These include the following: (a) initiation and maintenance of glycolysis, (b) cation pumping against electrochemical gradients, (c) synthesis of glutathione and other metabolites, (d) nucleotide catabolism reactions, (e) maintenance of Hb iron in its functional, reduced, ferrous state, (f) protection of enzymatic and structural proteins from oxidative denaturation, and (g) preservation of membrane phospholipid asymmetry.The structure of the RBC membrane (a thin layer that constitutes less than 0.1% of the cell thickness and only 1% of its weight) has been well elucidated in the past 35 years both from the normal and pathological metabolic points of view (1, 2) and, more recently, from a structural point of view via extensive proteomics mapping (3). Regarding the cytoplasmic content of the RBC, most studies have focused on a variety of rare ...

A review with >40 refs

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.

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).

The use of two types of peptide ligand libraries (PLL), containing hexapeptides terminating either with a primary amine or modified with a terminal carboxyl group, allowed the discovery and identification of a large number of previously unreported egg white proteins. Whereas the most comprehensive list up to date ( Mann, K. , Proteomics 2007, 7, 3558- 3568 ) tabulated 78 unique gene products, our findings have almost doubled that value to 148 unique protein species. From the initial nontreated egg, it was possible to find 41 protein species; the difference (107 proteins) was generated as a result of the use of PLLs from which a similar number of species (112 and 109, respectively) was evidenced. Of those, 35 proteins were the specific catch of the amino-terminus PLL, while 33 were uniquely captured by the carboxy-terminus PLL. While a number of these low-abundance proteins might have a biological role in maintaining the integrity of the egg white and protecting the yolk, others might be derived from decaying epithelial cells lining the oviduct and/or represent remnants of products from the magnum and eggshell membrane components secreted by the isthmus, which might ultimately be incorporated, even if in trace amounts, into the egg white. The list of egg white components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.