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PubMed2002

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation.

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.

Adenosine Triphosphatases↗
PubMedDate not supplied

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

B-Lymphocytes↗
PubMed2002

Peptide receptor imaging: advances in the diagnosis of pulmonary diseases.

Radiolabeled cell-surface peptide receptor-binding molecules are emerging as an important class of radiopharmaceuticals. Their binding to specific cell membrane receptors allows for noninvasive assessment of regional receptor proteomics in vivo. Information thus obtained can be used for diagnostic purposes and for predicting and monitoring response to treatment. This paradigm also applies to pulmonary diseases. In this review, available radiopharmaceuticals of great potential or already in clinical use for imaging of lung cancer, lung inflammation and infection and pulmonary embolism are discussed. In lung cancer, somatostatin receptor imaging by means of technetium-99m (99mTc)-octreotide scintigraphy has proven useful for characterizing malignancy in solitary pulmonary nodules. Additionally, several radiopharmaceuticals targeting tyrosine-kinase, e.g. 99mTc labeled epidermal growth factor and indium-111 (111In)-diethylene triamine penta-acetic acid-trastuzumab, or G-protein coupled receptors, e.g. 99mTc-bombesin, iodine-123-vasoactive intestinal peptide and 111In-tetraazacyclododecane tetra-acetic acid (DOTA)-cholecystokinine-B, are being explored for their diagnostic as well as treatment monitoring potential. With the purpose of better evaluating the source of pulmonary embolism, as well as to differentiate acute from chronic deep venous thrombosis, several radiolabeled peptides targeting the glycoprotein IIb/IIIa fibrinogen receptor found on activated platelets have been developed. Out of these, 99mTc-P280 is now approved by the US Food and Drug Administration for scintigraphic imaging of suspected acute venous thrombosis in the lower extremities of patients. In the field of lung inflammation and infection, non-specific 111In and 99mTc-human polyclonal immunoglobulins have been successfully used to identify the presence and extent of Pneumocystis carinii, cytomegalovirus, Mycobaterium avium and fungal infections in patients with HIV infection. The clinical role of other radiopharmaceuticals such as 99mTc-J001X, a nonpyrogenic acylated polygalactoside isolated from Klebsiella pneumoniae and binding with high affinity to CD11b and CD14 lipopolysaccharide receptors expressed on monocytes/macrophages, and 111In-octreotide, binding to up-regulated somatostatin receptors on activated lymphocytes needs to be further defined.

Carcinoma, Non-Small-Cell Lung↗
PubMed2003

Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response.

Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable isotope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the mixed protein samples by SDS-PAGE, followed by excision of the entire gel lane, and division of the lane into 14 gel regions. Regions were subjected to in-gel digestion, biotin affinity chromatography, and analysis by nano-scale microcapillary liquid chromatography coupled to tandem mass spectrometry. The novel (13)C-labeled ICAT reagents have identical elution profiles for labeled peptide pairs and broadly spread the distribution of labeled peptides during reversed-phase chromatography. A total of 560 proteins were identified and quantified, with 51 displaying more than 2-fold expression differences. In addition to some known proteins involved in salt stress, four RNA-binding proteins were found to be up-regulated by high salinity, suggesting that selective RNA export from the nucleus is important for the salt-stress response. Some proteins involved in amino acid synthesis, which have been observed to be up-regulated by amino acid starvation, were also found to increase their abundance on salt stress. These results indicate that salt stress and amino acid starvation cause overlapping cellular responses and are likely to be physiologically linked.

Amino Acid Sequence↗
PubMed2006

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

Amino Acid Sequence↗
PubMed2006

Differential expression of serum clusterin isoforms in colorectal cancer.

Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.

Biomarkers, Tumor↗
PubMed2004

RNA and DNA aptamers in cytomics analysis.

BACKGROUND: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. METHODS: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. RESULTS: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. CONCLUSIONS: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.

Animals↗
PubMed2003

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts.

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.

Amino Acid Sequence↗
PubMed2007

Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis.

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 microL) and a low concentration (5 x 10(-10) M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach.

Aluminum Oxide↗
PubMed2005

Theoretical investigations of prostatic acid phosphatase.

The phosphotyrosyl protein phosphatase activity of prostatic acid phosphatase (PAP) has been well established. It has also been suggested that PAP partly regulates the activity of growth factor receptors by dephosphorylating the autophosphorylysable tyrosines in them. We studied the binding of the peptides from epidermal growth factor receptor (EGFR) and its homolog (ErbB-2), corresponding to their autophosphorylation sites, to PAP using theoretical modeling and molecular dynamics (MD) simulation methods. Nine different peptides, each with a phosphotyrosine residue, were docked on human PAP. The binding energies of these peptide-PAP complexes were calculated theoretically and compared to experimentally obtained affinities. The peptide Ace--DNLpYYWD--NH2 from ErbB-2(1197-1203) showed the most favorable free energy of binding when estimated theoretically. The results demonstrate that the presence of another tyrosine residue proximate to C-terminal of autophosphorylysable Tyr enhances the binding affinity considerably. The presence of a bulky group instead prevents the binding, as is observed in case of peptide Ace--NLYpYWDQ--NH2 which failed to bind, both in theoretical calculations and experiments. Thus we demonstarted that PAP could potentially bind to EGFR and Erbb-2 and dephosphorylate them. Thus it could be involved in the regulation of the function of such receptors. In addition, complexes of a peptide from AngiotensinII and phosphotyrosine(pY) with human PAP were also modeled. The effects of different protonation states of the titratable active site residues on ligand (pY) binding have also been investigated. For a favorable binding His12 and Asp258 should be neutral, His257 should be positively charged and the phosphate group of the ligand should be in PO(4) (3-) state. Furthermore, the analysis of protein motion as observed during simulations suggests the loop-loop contact in the PAP dimer to be of importance in cooperativity.

Acid Phosphatase↗
PubMedDate not supplied

Global phosphoproteome of HT-29 human colon adenocarcinoma cells.

Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.

Adenocarcinoma↗
PubMed2005

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma.

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.

Animals↗
PubMed2005

Effect of chronic morphine exposure on the synaptic plasma-membrane subproteome of rats: a quantitative protein profiling study based on isotope-coded affinity tags and liquid chromatography/mass spectrometry.

The effect of chronic morphine exposure on the synaptic plasma-membrane subproteome in rats was studied by the isotope-coded affinity tag (ICAT) method coupled with capillary reversed-phase liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry. ICAT-labeled tryptic peptides of synaptic membrane proteins were successfully identified using tandem mass spectrometry in conjunction with protein database searching. Several important synaptic plasma-membrane proteins displayed significant regulation changes as a result of chronic morphine exposure in vivo. In particular, an integral membrane protein Na(+)/K+ ATPase (alpha-subunit) involved in regulation of the cell membrane potential by controlling sodium and potassium ion permeability was downregulated by 39 +/- 2%. This result was in excellent agreement with the reduction in electrogenic Na+, K+ pumping due to about 40% downregulation of Na(+)/K+ ATPase alpha3-isoform in myenteric S-neurons of morphine-exposed guinea-pigs measured by others via immunohistochemistry. The decrease in the abundance of non-erythroid alpha II-spectrin in the synaptic plasma-membrane fraction was also observed, which was hypothetically associated with the breakdown of the protein due to the upregulation of the proteolytic enzyme caspase-3 upon chronic morphine exposure.

Animals↗
PubMed2006

PIPE: a protein-protein interaction prediction engine based on the re-occurring short polypeptide sequences between known interacting protein pairs.

BACKGROUND: Identification of protein interaction networks has received considerable attention in the post-genomic era. The currently available biochemical approaches used to detect protein-protein interactions are all time and labour intensive. Consequently there is a growing need for the development of computational tools that are capable of effectively identifying such interactions. RESULTS: Here we explain the development and implementation of a novel Protein-Protein Interaction Prediction Engine termed PIPE. This tool is capable of predicting protein-protein interactions for any target pair of the yeast Saccharomyces cerevisiae proteins from their primary structure and without the need for any additional information or predictions about the proteins. PIPE showed a sensitivity of 61% for detecting any yeast protein interaction with 89% specificity and an overall accuracy of 75%. This rate of success is comparable to those associated with the most commonly used biochemical techniques. Using PIPE, we identified a novel interaction between YGL227W (vid30) and YMR135C (gid8) yeast proteins. This lead us to the identification of a novel yeast complex that here we term vid30 complex (vid30c). The observed interaction was confirmed by tandem affinity purification (TAP tag), verifying the ability of PIPE to predict novel protein-protein interactions. We then used PIPE analysis to investigate the internal architecture of vid30c. It appeared from PIPE analysis that vid30c may consist of a core and a secondary component. Generation of yeast gene deletion strains combined with TAP tagging analysis indicated that the deletion of a member of the core component interfered with the formation of vid30c, however, deletion of a member of the secondary component had little effect (if any) on the formation of vid30c. Also, PIPE can be used to analyse yeast proteins for which TAP tagging fails, thereby allowing us to predict protein interactions that are not included in genome-wide yeast TAP tagging projects. CONCLUSION: PIPE analysis can predict yeast protein-protein interactions. Also, PIPE analysis can be used to study the internal architecture of yeast protein complexes. The data also suggests that a finite set of short polypeptide signals seem to be responsible for the majority of the yeast protein-protein interactions.

Algorithms↗
PubMed2007

Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.

Defining protein complexes is critical to virtually all aspects of cell biology. Two recent affinity purification/mass spectrometry studies in Saccharomyces cerevisiae have vastly increased the available protein interaction data. The practical utility of such high throughput interaction sets, however, is substantially decreased by the presence of false positives. Here we created a novel probabilistic metric that takes advantage of the high density of these data, including both the presence and absence of individual associations, to provide a measure of the relative confidence of each potential protein-protein interaction. This analysis largely overcomes the noise inherent in high throughput immunoprecipitation experiments. For example, of the 12,122 binary interactions in the general repository of interaction data (BioGRID) derived from these two studies, we marked 7504 as being of substantially lower confidence. Additionally, applying our metric and a stringent cutoff we identified a set of 9074 interactions (including 4456 that were not among the 12,122 interactions) with accuracy comparable to that of conventional small scale methodologies. Finally we organized proteins into coherent multisubunit complexes using hierarchical clustering. This work thus provides a highly accurate physical interaction map of yeast in a format that is readily accessible to the biological community.

Cluster Analysis↗
PubMed2026

A Phosphoproteomic Platform Identifies Erythrocyte Membrane Protein Band 4.1-Like 3-Mediated Lipid Droplet Remodeling Linked to Liver Cancer Invasion and Migration.

Aberrant lipid metabolism is a hallmark of hepatocellular carcinoma (HCC), yet the regulatory mechanisms governing lipid droplet (LD) dynamics and their contribution to tumor progression remain poorly understood. Here, we developed an ultrasensitive phosphoproteomic platform using high-affinity HPDA@Ti4+ nanospheres to map LD-associated phosphorylation events across six HCC cell lines. By correlating phosphoproteomic signatures with LD morphology, we identified distinct regulatory signatures associated with LD size and abundance. Functional perturbation screens identified two distinct phosphoprotein modules controlling LD size: silencing SH3KBP1, SLK, EHD2, EPB41L3, and NEXN reduced LD size in Huh1 cells, whereas silencing CPD, BET1, UFL1, RRP1B, OGFR, and CD2BP2 enlarged LDs in Huh7 cells. Notably, we identified EPB41L3 as a critical metabolic-metastatic link; its loss decreased LD size and accelerated HCC migration and invasion, correlating with poor clinical prognosis. Crucially, we identified five key phosphorylation sites on EPB41L3 essential for its function; substituting these with alanine completely abolished its regulatory control over both LD size and HCC metastatic potential. Together, these findings delineate a phosphorylation-based regulatory network controlling the LD architecture and metastatic potential in HCC. Our study not only identifies potential therapeutic targets but also establishes a generalizable phosphoproteomic framework for interrogating lipid signaling in cancer metabolism.

Humans↗
PubMedDate not supplied

Riboproteomics of the hepatitis C virus internal ribosomal entry site.

Hepatitis C virus (HCV) protein translation is mediated by a cis-acting RNA, an internal ribosomal entry site (IRES), located in the 5' nontranslated region of the viral RNA. To examine proteins bound to the IRES, which could include proteins important for its function as well as potential drug targets, we used shotgun peptide sequencing to identify proteins in quadruplicate protein affinity extracts of lysed Huh7 cells, obtained using a biotinylated IRES. Twenty-six proteins bound the HCV IRES but not a reversed complementary sequence RNA or vector RNA controls. These included five ribosomal subunits, nine eukaryotic initiation factor 3 subunits, and novel interacting proteins such as the cytoskeletal-related proteins actin, FHOS (formin homologue overexpressed in spleen) and MIP-T3 (microtubule interacting protein that associates with TRAF3). Other novel HCV IRES-binding proteins included UNR (upstream of N-ras), UNR-interacting protein, and the RNA-binding proteins PAI-1 (plasminogen activator inhibitor-1) mRNA binding protein and Ewing sarcoma breakpoint 1 region protein EWS. A large set of additional proteins bound both the HCV IRES and a reversed complementary IRES sequence control, including the known HCV interactors PTB (polypyrimidine tract binding protein), the La autoantigen, and nucleolin. The discovery of these novel HCV IRES-binding proteins suggests links between IRES biology and the cytoskeleton, signal transduction, and other cellular functions.

Amino Acid Sequence↗
PubMedDate not supplied

Derivatized cellulose combined with MALDI-TOF MS: a new tool for serum protein profiling.

Providing a rapid and sensitive protein profiling method for biomarker discovery from a variety of biological samples is crucial for the introduction of new markers that improve cancer patient diagnosis at early tumor stages, thus increasing the chances of curative treatment. We report here the development and application of derivatized cellulose particles for selective serum protein profiling. For immobilized metal ion affinity chromatography (IMAC), cellulose was derivatized with glycidyl methacrylate (GMA) and subsequently with iminodiacetic acid (IDA). To investigate the application of this material for generating protein profiles of human serum samples, the serum samples were agitated with the derivatized cellulose particles to a suspension and incubated for 2 h at 30 degrees C. After washing, 1 microL of the IDA-Cu(2+)-cellulose suspension was applied directly onto a MALDI-target, mixed with sinapinic acid (SA) and analyzed with MALDI-TOF MS. Consistent serum specific data were obtained from aliquoted samples analyzed several times, indicating the reliability of the method. However, the serum fingerprints obtained proved to be specific for any given serum. The technique presented allows a high enrichment of sample on the developed target leading to a high sensitivity and reproducibility without depletion of albumin and immunoglobulin, and sample elution prior to MS-analysis. The study demonstrates for the first time that derivatized cellulose particles combined with MALDI-TOF MS represent a simple, economical, and rapid approach to generate serum protein profiles for biomarker identification.

Biomarkers↗