Carotid atherosclerotic plaques: proteomics study after a low-abundance protein enrichment step.

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin ß-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.

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.

Culture of human cells and synthesis of extracellular matrix on materials compatible with direct analysis by mass spectrometry.

The extracellular matrix (ECM) is a complex three-dimensional network of macromolecules synthesized by cells and is essential for the structure and the function of a tissue. The aim of our approach was to propose a surface allowing cell culture and subsequent analysis of ECM produced by cells directly on materials compatible with Surface Enhanced Laser Desorption Ionization-Time Of Flight (SELDI-TOF) mass spectrometry on a 96-well format. Surfaces were made of aluminium and spots of 2 mm in diameter were covered with specific chemical groups (silica, C(6) and C(12) alkyl groups, carboxyl, quaternary amine, or nitrilotriacetic acid groups). We found that among the chemically modified aluminium spots, only silica groups allowed the culture of human vascular cells. The wettability was an essential parameter for cell culture on the surfaces. Indeed, cells could only be cultured on surfaces presenting a moderate wettability with water contact angles of ca. 60 degrees. Then, by treatment of confluent cells with detergents (Triton X100 and deoxycholate), we were able to obtain ECM on the surfaces that were subsequently analyzed using a mass spectrometer, which is currently impossible with any type of cell culture system. As an example, the analysis of ECM from human vascular smooth muscle cells (hVSMCs) and human umbilical vein endothelial cells (HUVECs) appeared to be reproducible and evidenced different ECM patterns from the two cell types. Applications based on these materials can be proposed for biomarker discovery or characterization of cells for biomedical/diagnostic purposes.

Ovine serum biomarkers of early and late phase scrapie.

BACKGROUND: Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no ante-mortem diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant. METHODS: In this study we used the SELDI-TOF MS technology to analyze a large number of serum samples from control sheep and animals with early phase or late phase scrapie. A few potential low molecular weight biomarkers were selected by statistical methods and, after a training analysis, a protein signature pattern, which discriminates between early phase scrapie samples and control sera was identified. RESULTS: The combination of early phase biomarkers showed a sensitivity of 87% and specificity of 90% for all studied sheep in the early stage of the disease. One of these potential biomarkers was identified and validated in a SELDI-TOF MS kinetic study of sera from Syrian hamsters infected by scrapie, by western blot analysis and ELISA quantitation. CONCLUSIONS: Differential protein expression profiling allows establishing a TSE diagnostic in scrapie sheep, in the early phase of the disease. Some proteic differences observed in scrapie sheep exist in infected hamsters. Further studies are being performed to identify all the discriminant biomarkers of interest and to test our potential markers in a new cohort of animals.

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.

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.

Performance of combinatorial peptide libraries in capturing the low-abundance proteome of red blood cells. 2. Behavior of resins containing individual amino acids.

UNLABELLED: Sixteen different amino acids (Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val) have been separately linked to chromatographic beads and used for studying the mechanism of binding of such baits to proteins, as represented by the cytoplasmic proteome of the human red blood cell (RBC). The 16 different amino acid columns were confronted with equal amounts of RBC lysate, washed to remove unbound material, and eluted with denaturing agents. All eluates were analyzed by nanoLC-MS/MS. THE RESULTS: there appears to be a dichotomy between a class of "Grand Catchers" (Arg, His, Ile, Lys, Phe, Trp, Tyr, Val), all able to bind from 330 up to 441 unique gene products, and the "Petite Catchers" (Asn, Asp, Gln, Glu, Gly, Pro, Ser, Thr), that bind in general half as much, with the notable exception of Glu that under the described conditions seems to bind only traces of proteins. By comparing homogeneous classes of amino acids (e.g., the basic, the hydrophobic aromatic, the neutral hydrophilic, etc.), it is found that, in general, more than half as many proteins are held in common among the members of each family. In a 16-way comparison, 72 proteins (less than 10% of the total amount, which amounts to 800 unique, nonredundant, identified proteins) appear to be the common catch of all 16 amino acids, suggesting that such proteins might have either multiple binding sites or general motifs recognized by any generic bait. By far, it would appear that the strongest interactions and thus the strongest catches occur with the three aromatic moieties of Phe, Trp, and Tyr, all able to capture a practically identical number of proteins. Ionic interactions, which in principle should be the strongest ones, appear to behave in a peculiar way: they are quite strong with the three basic amino acids (Arg, His, Lys) but almost inexistent with their acidic counterparts. This suggests a peculiar mechanism of interaction: upon formation of the ion pair, the linkage between the protein and the bait is stabilized by the hydrophobicity of the underlying chain (e.g., a butyl in the case of Lys).

Reducing protein concentration range of biological samples using solid-phase ligand libraries.

The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called "top down" or "bottom up" approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.

Depletion of proBNP1-108 in patients with heart failure prevents cross-reactivity with natriuretic peptides.

BACKGROUND: After synthesis by cardiomyocytes, precursor proBNP1-108 is cleaved into NT-proBNP and BNP. Recently, cross-reactivity between these assays was discussed. The aim of this study was to characterize the cross-reactivities, through a new biochemical innovative approach consisting in the total depletion of the circulating proBNP1-108 in patients with heart failure (HF). METHODS: This prospective study included 180 patients with chronic HF. BNP and NT-proBNP were dosed with commercial kits. ProBNP1-108 was determined using an ELISA research assay specific to the precursor. ProBNP1-108 depletion was performed by immunocapture with a specific antibody targeting exclusively the ProBNP1-108 hinge region. ProBNP1-108, BNP and NT-proBNP levels were determined before and after depletion using this process in HF patients. RESULTS: Mean age was 74.34 +/-12.5 y, and 69% of patients were males. NYHA classes II and III were the most frequent (32% and 45% respectively). Before depletion, ProBNP1-108, NT-proBNP and BNP levels were 316.8+/-265.9 pg/ml; 6,054.0+/-11,539 pg/ml and 684.3+/-82.1 pg/ml respectively, and were closely correlated with NHYA classes. After immuno-depletion, proBNP1-108 was decreased in mean by 96% (p<0.0001), BNP by 53% (p<0.0001) and NT-proBNP by 5%. The relationship between BNP or NT-proBNP and NHYA classes remained unchanged. CONCLUSION: Current BNP and NT-proBNP assays measured as well proBNP molecule. This cross reactivity percentage has been controversial. Thanks to the removal of circulating proBNP1-108 with our immunodepletion process, we are now able to assess the remaining "true" BNP and NT-proBNP molecules and further evaluate their clinical relevance.

Solid-phase fractionation strategies applied to proteomics investigations.

Methods for protein fractionation in the proteomics investigation field are relatively numerous. They apply to the prefractionation of the sample to obtain less complex protein mixtures for an easier analysis; they are also used as a means to evidence specific proteins or protein classes otherwise impossible to detect. They involve depletion of high-abundance proteins suppressing the signal of dilute species; they are also capable to enhance the detectability of low-abundance species while concomitantly decreasing the concentration of abundant proteins such as albumin in serum and hemoglobin in red blood cell lysates. Fractionation of proteomes is also used for the isolation of targeted species that are selected for their different expression under certain pathological conditions and that are detected by mass spectrometry. Two unconventional methods of large interest in proteomics due to the low level of protein redundancy between fractions are also reported.All these methods are reviewed and detailed method given to allow specialists of proteomics investigation to access selected separation methods generally dispersed on different technical reviews or books.

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.

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.

pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries.

The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods. In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions. What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation. A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation.

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.

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.

Evaluation of a standardized method of protein purification and identification after discovery by mass spectrometry.

Identification of unknown proteins subsequent to a mass spectrometry signal is still a serious obstacle in the discovery of relevant biomarkers of diagnostic interest. In this report the evaluation of a rational process under optimized conditions is described for three unknown proteins representing important targets in their field of investigation. The process, involving few dozens of chromatographic sorbents and two buffers, allowed identifying prothrombin fragment 1, a minor glycoprotein of human serum with inhibitory activity associated with pathogenesis of calcium oxalate stones. The same technology demonstrated its efficiency for the separation of a recombinantly expressed yeast transcription factor in Escherichia coli with subsequent formal identification. In addition, a DNA-binding protein from the gastric pathogen Helicobacter pylori has been separated by the same technology and formally identified. The reported data show that the method is reliable and easily applicable to a large variety of cases with a standardized approach. Identity coverage and relative abundance after purification and removal of critical protein impurities are reported. Examples of protein isolation/identification are described, namely PTF1, recombinant YAP-1 transcription factor from E. coli and DNA-binding protein HU from H. pylori. Isolated proteins were pure enough for the purpose of formal identification by either peptide mass fingerprinting or sequencing.

A pI-based protein fractionation method using solid-state buffers.

The analysis of very complex proteomes is dependent on efficient fractionation methods with low level of carry over from fraction to fraction. Among various possibilities the separation by ranges of isoelectric points for further analysis appears as attractive, but current methods involving an electrically driven migration in the presence of ampholyte carriers are not exempt of technical complications. In the present work a new separation concept is described involving the use of so-called solid-state buffers, in association with ion exchangers, to separate protein categories of different pI ranges with a low level of protein overlapping. Resin blends packed in separated columns are used under a cascade configuration of increasing or decreasing pH and, once proteins of different pI are adsorbed by individual resin blends, the columns are dissociated. From each column protein mixtures corresponding to a given pI range are collected by competitive desorption with salts so as to be ready for proteomic analysis. The process is rapid and does not involve electrical fields nor addition of carrier ampholyte material. The presence of potassium chloride during the separation prevents protein precipitation at the vicinity of their isoelectric points. The fractions thus obtained can be used for two dimensional electrophoresis and mass spectrometry analysis after the removal of salts.

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.

Protocol for the purification of proteins from biological extracts for identification by mass spectrometry.

When a protein signal is selected by mass spectrometry as being a potential biomarker, it is necessary to formally identify it. This process involves separation of the protein in question and its identification by either peptide fingerprinting or tandem mass spectrometry sequencing. In the following pages, a simple and rapid protocol is described. Basically, the protocol consists of an initial rational selection of a few sorbents followed by alignment of these as a series of columns to obtain the purified target protein. This preparation is then submitted to electrophoresis, the band is excised and the trypsin digest is analyzed by either mass spectrometry (mass fingerprinting approach) or by LC-MS/MS (sequencing). The development of the process takes only a few days. Experimental data for the isolation and identification of proteins are discussed and two examples are shown.

Capturing and amplifying impurities from purified recombinant monoclonal antibodies via peptide library beads: a proteomic study.

Capture and amplification of low-level contaminants in purified preparations of recombinant DNA products is described here in the case of mAb meant for human consumption. Such a process is based on treatment with a vastly heterogeneous ligand library composed of hexapeptides bound to a polyhydroxymethacrylate resin. Upon this treatment, a protein solution is recovered with "normalized" relative concentration ratios, in which high-abundance proteins are strongly reduced and rare proteins are highly concentrated. Upon 2-D map analysis, the relatively few spots present in control monoclonals were seen to increase in number, reaching >100 visible polypeptide chains in the pI/M(r) plane. Most of these newly emerged spots were subjected to MS analysis and were found to be composed mainly of three classes of proteins: those derived from proteins present in the culture broth (notably albumin and transferrin), fragments of the desired final product, covering M(r) ranges from as low as 5 up to 45 kDa and some aggregates of light and heavy chains of Igs (mostly dimers and trimers). This ligand library thus appears to be a formidable tool for exploring and bringing to the limelight the "hidden proteome".