Luc Guerrier

Extensive Analysis of the Cytoplasmic Proteome of Human Erythrocytes Using the Peptide Ligand Library Technology and Advanced Mass Spectrometry

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.

Exploring the chicken egg white proteome with combinatorial peptide ligand libraries.

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.

In-depth Exploration of Cerebrospinal Fluid by Combining Peptide Ligand Library Treatment and Label-free Protein Quantification

Cerebrospinal fluid (CSF) is the biological fluid in closest contact with the brain and thus contains proteins of neural cell origin. Hence, CSF is a biochemical window into the brain and is particularly attractive for the search for biomarkers of neurological diseases. However, as in the case of other biological fluids, one of the main analytical challenges in proteomic characterization of the CSF is the very wide concentration range of proteins, largely exceeding the dynamic range of current analytical approaches. Here, we used the combinatorial peptide ligand library technology (ProteoMiner) to reduce the dynamic range of protein concentration in CSF and unmask previously undetected proteins by nano-LC-MS/MS analysis on an LTQ-Orbitrap mass spectrometer. This method was first applied on a large pool of CSF from different sources with the aim to better characterize the protein content of this fluid, especially for the low abundance components. We were able to identify 1212 proteins in CSF, and among these, 745 were only detected after peptide library treatment. However, additional difficulties for clinical studies of CSF are the low protein concentration of this fluid and the low volumes typically obtained after lumbar puncture, precluding the conventional use of ProteoMiner with large volume columns for treatment of patient samples. The method has thus been optimized to be compatible with low volume samples. We could show that the treatment is still efficient with this miniaturized protocol and that the dynamic range of protein concentration is actually reduced even with small amounts of beads, leading to an increase of more than 100% of the number of identified proteins in one LC-MS/MS run. Moreover, using a dedicated bioinformatics analytical work flow, we found that the method is reproducible and applicable for label-free quantification of series of samples processed in parallel.

Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library.

Deciphering the protein composition of complex extracts or discovering biologically relevant polypeptides is frequently hindered by large dynamic concentration ranges. The presence of high-abundance proteins suppresses the signal of low-abundance ones, and the most rare proteins are frequently below the sensitivity of available analytical methods. This protocol addresses this problem by using highly diversified hexapeptide ligand libraries for capturing proteins. A protein extract is mixed with the library and because the library has equal amounts of each ligand, theoretically the maximum amount of each protein bound is the same. Under overloading conditions this has the effect of diluting those proteins present in excess of the ligand concentration and concentrating those of relatively lower abundance. Unbound components are washed out and captured species are finally desorbed. The entire sample treatment process takes about half a day and yields a protein solution that could be used as such for mass spectrometry investigations.

Chicken egg yolk cytoplasmic proteome, mined via combinatorial peptide ligand libraries.

The use of combinatorial peptide ligand libraries (CPLLs), containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, or with a terminal tertiary amine, allowed discovering and identifying a large number of previously unreported egg yolk proteins. Whereas the most comprehensive list up to date [K. Mann, M. Mann, Proteomics, 8 (2008) 178-191] tabulated about 115 unique gene products in the yolk plasma, our findings have more than doubled this value to 255 unique protein species. From the initial non-treated egg yolk it was possible to find 49 protein species; the difference was generated thanks to the use of the three combined CPLLs. The aberrant behaviour of some proteins, upon treatment via the CPLL method, such as proteins that do not interact with the library, is discussed and evaluated. Simplified elution protocols from the CPLL beads are taken into consideration, of which direct elution in a single step via sodium dodecyl sulphate desorption seems to be quite promising. Alternative methods are suggested. The list of egg yolk 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.

High-performance protein separations with novel strong ion exchangers

Abstract In this paper, properties of new ion exchangers specifically designed for protein separations are reported. These sorbents are constituted of two main parts: a rigid, porous polystyrene-silica composite material which forms a rigid skeleton and a soft hydrogel bringing strong ionic groups. The later is regularly distributed inside the pores of the skeleton. Characterization of these materials was performed by measuring dynamic sorption capacity, resolving power, separation efficiency and protein recovery. These studies were done using various known proteins and protein mixtures. Some comparisons have been made with commercially available ion exchangers also designed for protein separations.

Interaction among proteins and peptide libraries in proteome analysis: pH involvement for a larger capture of species.

When capturing proteins via combinatorial peptide ligand libraries, a method well known for drastically reducing the concentration of high-abundance proteins and substantially magnifying the signal of low-abundance species, thus leading to the discovery of a large number of proteins previously undetected in proteomes, we had constantly noticed that there would be a loss of species initially present in the untreated sample, to the tune of 5%, up to 15% in some cases. Such losses are a nuisance and hamper to some extent the unique performance of the method. In order to verify if such losses could be reduced and also to understand some mechanisms of the capture process, we introduce here an important variant to the capture operation, up to the present carried out in physiological saline at pH 7.2. In this novel protocol, the binding step is conducted at three different pH values, namely the standard one at pH 7.2, plus two additional processes, at acidic (pH 4.0) and alkaline (pH 9.3) pH values. Indeed the capture process is more extensive, with a number of additional species captured at the two pH extremes in sera and other proteomes. Interestingly, at pH 4.0 newly detected proteins were mostly acidic, while at the alkaline pH additional protein species were more evenly distributed throughout the pI range towards the alkaline area. The role of pH in the complex mechanism of binding among the hexapeptide library and the various proteomes being analyzed is discussed and evaluated. Due to significant changes in protein patterns with pH, recommendations are thus made to increase the possibility to find additional gene products illustrated by two examples (snake venom and leaf protein extract). Keeping under control the environmental pH when facing reproducibility studies or for comparative proteomics profiling is also a general rule suggested by this study.

Performance of Combinatorial Peptide Libraries in Capturing the Low-Abundance Proteome of Red Blood Cells. 1. Behavior of Mono- to Hexapeptides

UNLABELLED For a better understanding of the behavior of solid-phase combinatorial peptide ligands for capturing the red blood cell low-abundance soluble proteome, combinatorial peptides of different lengths from a single amino acid up to a hexapeptide were evaluated. A red blood cell lysate (6 g total protein) was loaded in a cascade fashion to the six columns, which were individually eluted with 8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (v/w), and 50 mM citric acid. Each eluate was analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional maps, and nanoLC-MS/MS. THE RESULTS mixed beads with a single amino acid attached showed the capture of a non-negligible portion of the proteome. A progressive increasing of the length of the peptide bait enlarges the pool of captured proteins. Above a length of four amino acids, a plateau is progressively reached, suggesting that not much could be gained with baits longer than six amino acids. Interestingly, whereas the beads laden with a single amino acid seem to be able to capture large-size proteins (>40 kDa), beads with progressively longer peptides capture additional proteins in the smaller size range (10-50 kDa). This suggests that interactions already begin with a single amino acid, but selectivity requires baits of proper length, at least above four amino acids. Plain beads, with a spacer arm carrying a primary amino terminal group for anchoring the baits, are essentially unable to capture proteins, suggesting that the peptide baits do not act by a mechanism of ion exchange but rather via a complex mixed mode, yielding a specific capture.

Preparative high-performance liquid chromatographic separation of proteins with HyperD ion-exchange supports

HyperD ion-exchange media combine the mechanical strength of a rigid polystyrene-mineral composite skeleton with the high protein-binding capacity of a three-dimensional soft gel located inside the skeleton. The skeleton solid matrix is completely filled with functionalized, highly hydrophilic, chemically stable ion-exchange hydrogels. These materials gave very efficient columns for protein separation with superior dynamic capacity, high resolving power and excellent protein recovery. Various protein mixtures were used to study the chromatographic performance of these new stationary phases. Comparisons between different particle size packing materials demonstrated the potential of this ion-exchange material for use on a large scale.

Solid-phase hexapeptide ligand libraries open up new perspectives in the discovery of biomarkers in human plasma

BACKGROUND Pre-treatment of plasma with hexapeptide ligand libraries prior to proteomic analysis is well documented. However, the maintenance of biomarker abundance throughout the different pre-analytical steps is required for a potential application of differential proteomics in clinical studies. METHODS We combined the use of an amino-terminal hexapeptide ligand library and its carboxyl-terminal version with a sequential elution strategy of the proteins/peptides bound to the beads, followed by either mass spectrometry or 2D electrophoresis analyses. RESULTS We show the maintenance of C-reactive protein abundance (a marker of inflammation) throughout the process (including hexapeptide bead treatment and proteomic analysis) in patients presenting high and low levels of this protein. In parallel, we assessed the contribution of this workflow to increasing the number of potential biomarkers detected and its suitability for a clinical study on approximately a hundred samples, as well as the reproducibility of the process. CONCLUSIONS Pre-treatment with hexapeptide ligand libraries opens up new perspectives in the discovery of biomarkers in human plasma by improving the detection of new species while maintaining their original differential abundance. This approach is also suitable for an application in a clinical proteomic study of at least 100 samples.