Proteomic analysis of human pleural effusion

Pleural effusion, an accumulation of pleural fluid, contains proteins originating from plasma filtrate and, especially when tissues are damaged, parenchymal interstitial spaces of lungs and/or other organs. This report presents data of the first global proteomic analysis of human pleural effusion. A composite sample was prepared by pooling pleural effusions from seven lung adenocarcinoma patients. Two-dimensional gel electrophoresis analysis of the composite sample revealed 472 silver-stained spots. 242 selected gel spots were subjected to protein identification by in-gel digestion, liquid chromatography-tandem mass spectrometry, and sequence database search. 44 proteins were identified with higher confidence levels (at least two unique peptide sequences matched), while 161 other proteins were identified at the minimal confidence level (only one unique peptide sequence matched). The data provide fundamental information on the composition of protein contents in human pleural effusion. Among these 44 proteins that were identified with higher confidence levels, 7 proteins, retinoblastoma binding protein 7, synaptic vesicle membrane protein, corticosteroid binding globulin precursor, PR-domain containing protein 11, envelope glycoprotein, MSIP043 protein, and titin have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins could have originated from parenchymal interstitial spaces and represent potential candidates of useful biomarkers that could not be readily detected in plasma but in pleural effusion. Retinoblastoma binding protein 7 is of special interest since it may play a role in the regulation of cell proliferation and differentiation.     

Proteome analysis of Myxococcus xanthus by off-line two-dimensional chromatographic separation using monolithic poly-(styrene-divinylbenzene) columns combined with ion-trap tandem mass spectrometry

Myxobacteria are potent producers of secondary metabolites exhibiting diverse biological activities and pharmacological potential. The proteome of Myxococcus xanthus DK1622 was characterized by two-dimensional chromatographic separation of tryptic peptides from a lysate followed by tandem mass spectrometric identification. The high degree of orthogonality of the separation system employing polymer-based strong cation-exchange and monolithic reversed-phase stationary phases was clearly demonstrated. Upon automated database searching, 1312 unique peptides were identified, which were associated with 631 unique proteins. High-molecular polyketide synthetases and nonribosomal peptide synthetases, known to be involved in the biosynthesis of various secondary metabolites, were readily detected. Besides the identification of gene products associated with the production of known secondary metabolites, proteins could also be identified for six gene clusters, for which no biosynthetic product has been known so far.     

The art of observing rare protein species in proteomes with peptide ligand libraries

The present review deals with the use of combinatorial ligand libraries, composed by hexapeptides, in the capture and concentration of the low‐abundance proteome. This method, first reported in 2005, is compared with other current methodologies aimed at exploring the “deep proteome”, such as: depletion of high‐abundance proteins (especially in sera and cerebrospinal fluid) by individual or combined antibodies (up to 20 against the most abundant species); narrow (1‐pH‐unit) IPGs (zoom gels) and prefractionation with multicompartment electrolyzers or with Off‐Gel electrophoresis. The physico‐chemical properties of the hexapeptide library are also explored, namely in assessing the proper length of the baits and the behavior of shorter oligopeptides, down to capture elicited by single amino acids. A number of examples on the use of this library is given, such as the analysis of biological fluids (human sera, urine, bile, cerebrospinal fluid) and of cell lysates (platelets, red blood cells). In all cases, it was possible to detected from three to five times as many proteins as compared to control, untreated samples. Perhaps the most spectacular results were obtained with the erythrocyte proteome, where 1570 proteins could be identified in the “minority” proteome, representing only 2% of the total cell lysate. Another interesting area of application regards the concentration and detection of trace impurities contaminanting r‐DNA proteins meant for human consumption: several host proteins, never reported before, could be revealed for the first time. Other nonhuman samples are currently under investigation, such as egg‐white (where no less than 148 unique gene products could be identified), egg yolk (with 255 unique species) and latex from Hevea brasiliensis. It is anticipated that the ligand library could be a most useful tool for detecting biomarkers for different pathologies and for drug treatment. As a future outlook, one could envision the synthesis of specialized libraries able to capture given classes of proteins (e.g., phospho‐ and glyco‐proteins). Additionally, the library could be used in association with other techniques currently in vogue, such as zoom gels, Off‐Gels, and the like.     

Harry Belafonte and the secret proteome of coconut milk

The proteome of coconut milk has been extensively mapped via capture at three pH values with combinatorial peptide ligand libraries (CPLL). A grand total of 307 unique gene products could be listed, 200 discovered via CPLL capture, 137 detected in the control, untreated material and 30 species in common between the two sets of data. This is by far the most extensive mapping of coconut milk, in which, up to the present, only a dozen proteins were known, those belonging to the high- to very-high abundance class. The database of coconut contains only 106 proteins: of those, only six are listed in our table. The vast majority of the classified proteins, thus, has been identified only by homologies with sequences deposited in the general viridiplantae database. This unique set of data could be the starting point for nutritionists and researchers involved in nutraceutics for enucleating some proteins responsible for some of the unique beneficial health effects attributed to coconut milk.     

In-Depth Characterization of Sheep (Ovis aries) Milk Whey Proteome and Comparison with Cow (Bos taurus)

An in-depth proteomic study of sheep milk whey is reported and compared to the data available in the literature for the cow whey proteome. A combinatorial peptide ligand library kit (ProteoMiner) was used to normalize protein abundance in the sheep whey proteome followed by an in-gel digest of a 1D-PAGE display and an in-solution digestion followed by OFFGEL isoelectric focusing fractionation. The peptide fractions obtained were then analyzed by LC-MS/MS. This enabled identification of 669 proteins in sheep whey that, to our knowledge, is the largest inventory of sheep whey proteins identified to date. A comprehensive list of cow whey proteins currently available in the literature (783 proteins from unique genes) was assembled and compared to the sheep whey proteome data obtained in this study (606 proteins from unique genes). This comparison revealed that while the 233 proteins shared by the two species were significantly enriched for immune and inflammatory responses in gene ontology analysis, proteins only found in sheep whey in this study were identified that take part in both cellular development and immune responses, whereas proteins only found in cow whey in this study were identified to be associated with metabolism and cellular growth.     

Use of peptide analogue diversity library beads for increased depth of proteomic analysis: application to cerebrospinal fluid

Biological samples can contain proteins with concentrations that span more than 10 orders of magnitude. Given the limited dynamic range of analysis methods, observation of proteins present at the lower concentrations requires depletion of high-abundance proteins, or other means of reducing the dynamic range of concentrations. Hexapeptide diversity library beads have been used to bind proteins in a complex sample up to a given saturation limit, effectively truncating the maximum concentration of proteins at a desired level. To avoid the potential problem of susceptibility of the hexapeptides to cleavage by proteases in the sample and/or bacterial degradation, peptide analogues that exhibit similar binding characteristics to peptides can be used in place of peptides. We report here the use of hexameric peptoid diversity library beads to reduce the dynamic range of protein concentrations in human cerebrospinal fluid (CSF). Using this method in conjunction with 2D LC/MS/MS analyses, we identified 200 unique proteins, about twice the number identified in untreated CSF.     

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.     

Multidimensional proteome analysis of human mammary epithelial cells

Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with the separation of tryptically digested peptides using a combination of strong cation exchange and high resolution, reversed phase capillary chromatography to identify proteins extracted from human mammary epithelial cells (HMECs). In addition to conventional conservative criteria for protein identifications, the confidence levels were additionally increased through the use of peptide normalized elution times (NET) for the liquid chromatographic separation step. The combined approach resulted in a total of 5838 unique peptides identified covering 1574 different proteins with an estimated 4% gene coverage of the human genome, as annotated by the National Center for Biotechnology Information (NCBI). This database provides a baseline for comparison against variations in other genetically and environmentally perturbed systems. Proteins identified were categorized based upon intracellular location and biological process with the identification of numerous receptors, regulatory proteins, and extracellular proteins, demonstrating the usefulness of this application in the global analysis of human cells for future comparative studies.     

Cibacron Blue and proteomics: the mystery of the platoon missing in action

The use of Cibacron Blue columns (HiTrapBlue) in proteome analysis for removal of plasma albumin, for facilitating biomarker discovery, has not borne any fruit. In fact, the visibility of low-abundance proteins was obscured. It is here reported that, upon albumin sequestering from plasma, there is adsorption, via hydrophobic interaction, of a substantial number of plasma proteins, which are lost for subsequent analysis if the blue resin is eluted via an ion shock (2 M NaCl) or with a somewhat more robust eluant (5 M urea, 2 M thiourea, 2% CHAPS, 2% sulphobetain 3-10) as recommended by manufacturers. Such treatments, in fact, release at most 25 to 30 unique gene products, including albumin. If, however, the Affigel-Blue resin, after elution with either of the two above eluants, is further eluted with boiling 4% SDS in 25 mM DTT, all the missing proteins (amounting to at least 112 unique species) are desorbed and biomarker analysis can be conducted in a correct way. It is also suggested that such blue-resin treatment could be coupled to ProteoMiner adsorption, this coupled treatment further enhancing the chances of success for discovery of low-abundance proteins.     

Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS

Saliva is a readily available body fluid with great diagnostic potential. The foundation for saliva-based diagnostics, however, is the development of a complete catalog of secreted and "leaked" proteins detectable in saliva. By employing a capillary isoelectric focusing-based multidimensional separation platform coupled with electrospray ionization tandem mass spectrometry (MS), a total of 5338 distinct peptides were sequenced, leading to the identification of 1381 distinct proteins. A search of bacterial protein sequences also identified many peptides unique to several organisms and unique to the NCBI nonredundant database. To the best of our knowledge, this proteome study represents the largest catalog of proteins measured from a single saliva sample to date. Data analysis was performed on individual MS/MS spectra using the highly specific peptide identification algorithm, OMSSA. Searches were conducted against a decoyed SwissProt human database to control the false-positive rate at 1%. Furthermore, the well-curated SwissProt sequences represent perhaps the least redundant human protein sequence database (12,484 records versus the 50,009 records found in the International Protein Index human database), therefore minimizing multiple protein inferences from single peptides. This combined bioanalytical and bioinformatic approach has established a solid foundation for building up the human salivary proteome for the realization of the diagnostic potential of saliva.     

Exploring the platelet proteome via combinatorial, hexapeptide ligand libraries

A combinatorial ligand library, composed of millions of diverse hexapeptide baits, able to capture and concentrate the "low-abundance" proteome while drastically cutting the concentration of the most abundant species, has been applied to the exploration of the soluble platelet proteome. Mass spectrometry analysis of untreated and library-treated platelets has resulted in the identification of 435 unique gene products. Of those, 147 entries (35% of the total) have not been described among the list of >1100 proteins in proteomic platelet investigations reported before. In addition, the analysis of excised spots from two-dimensional electrophoresis analysis allowed 57 other proteins to be added that were not found in LC-MS analysis, 33 of them not described before in proteomics studies, bringing the total number of new gene products to 180. Thus, the present data add a non-negligible number of species for continuing the "cartography" of the proteomic asset of platelets, in view of completing the mapping procedure for a deeper understanding of the physiology and pathology of this blood cell. Because the capturing process is performed under physiological conditions, by exploiting, for binding to the combinatorial library, the native protein configuration, the described technique is not adapted to capture highly hydrophobic proteins, which need strong denaturing and solubilizing conditions that are incompatible with our working procedure. Thus, our list reports essentially hydrophilic proteins, with negative GRAVY indexes.     

Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns

It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.     

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.     

The proteome buccaneers: how to unearth your treasure chest via combinatorial peptide ligand libraries

The latest advances in combinatorial peptide ligand libraries, with their unique performance in discovering low-abundance species in proteomes, are reviewed here. Explanations of mechanism, potential applications, capture of proteomes at different pH values to enhance the total catch and quantitative elutions, such as boiling in the presence of 5% sodium dodecyl sulfate and 3% dithiothreitol are included. The reproducibility of protein capture among different experiments with the same batch of beads or with different batches is also reported to be very high, with coefficient of variations in the order of 10–20%. Miniaturized operations, consisting of capture with as little as 20 or even 5 µl of peptide beads are reported, thus demonstrating that the described technology could be exploited for routine biomarker discovery in a biomedical environment. Finally, it is shown that the signal of captured proteins is linear over approximately three orders of magnitude, ranging from nM to µM, thus ensuring that differential quantitative proteomics for biomarker discovery can be fully implemented, providing species do not saturate their ligands.     

Looking deep inside: detection of low-abundance proteins in leaf extracts of Arabidopsis and phloem exudates of pumpkin

The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome.     

Proteomic identification of plasma biomarkers in uterine leiomyoma

Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of uterine leiomyomas. In the present study, plasma samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 20 proteins have been firmly identified representing 13 unique gene products. These proteins mainly functioned in transportation (such as apolipoprotein A-I) and coagulation (such as fibrinogen gamma chain). Additionally, our quantitative proteomic approach has identified numerous previous reported plasma markers of uterine leiomyomas such as alpha-1-antitrypsin. On the contrary, we have presented several putative uterine leiomyomas biomarkers including afamin, apolipoprotein A-I, carbonic anhydrase 1, fibrinogen beta chain, fibrinogen gamma chain, gelsolin, hemopexin, leucine-rich alpha-2-glycoprotein, serotransferrin and vitamin D-binding protein which have not been reported and may be associated with the progression and development of the disease. In summary, we report a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers for uterine leiomyomas. The potential of utilizing these markers for screening and treating uterine leiomyomas warrants further investigations.