In-gel Trypsin Digestion Research Articles

Activated Inositol 1,4,5-Trisphosphate Receptors Are Modified by Homogeneous Lys-48- and Lys-63-linked Ubiquitin Chains, but Only Lys-48-linked Chains Are Required for Degradation

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP(3) and Ca(2+)-gated Ca(2+) channels. Endogenous IP(3)Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP(3)R1 in αT3-1 pituitary gonadotrophs and found that IP(3)R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D. A., Kubota, K., Kirkpatrick, D. S., Alzayady, K. J., Gygi, S. P., and Wojcikiewicz, R. J. H. (2008) J. Biol. Chem. 283, 35319-35328). Here, we have extended these studies to determine whether IP(3)R2 and IP(3)R3 are similarly modified and if ubiquitination is cell type-dependent. Using mass spectrometry and linkage-specific ubiquitin antibodies, we found that all IP(3)R types are subject to ubiquitination at approximately the same locations and that, independent of cell type, IP(3)Rs are modified by monoubiquitin and Lys-48- and Lys-63-linked ubiquitin chains, although in differing proportions. Remarkably, the attached Lys-48- and Lys-63-linked ubiquitin chains are homogeneous and are segregated to separate IP(3)R subunits, and Lys-48-linked ubiquitin chains, but not Lys-63-linked chains, are required for IP(3)R degradation. Together, these data provide unique insight into the complexities of ubiquitination of an endogenous ubiquitin-proteasome pathway substrate in unperturbed mammalian cells. Importantly, although Lys-48-linked ubiquitin chains appear to trigger proteasomal degradation, the presence of Lys-63-linked ubiquitin chains suggests that ubiquitination of IP(3)Rs may have physiological consequences beyond signaling for degradation.

Abstract P6-04-11: Quantitative Differences among Spectral Counts Based on LC-MS/MS Spectra Accurately Cluster Basal, Her2-Overexpressing and Luminal A Breast Carcinomas

Abstract Characterization of breast cancer subtypes at the protein-level is largely unexplored and is a powerful approach to identify novel biomarkers that may have diagnostic and prognostic utility. Shotgun proteomics analyzes mixtures at the peptide-level to generate MS/MS spectra that are then used to identify the peptides and proteins from which they were derived. We developed a method in our laboratory utilizing SDS-PAGE followed by in-gel digestion coupled with reversed-phase (RP) liquid chromatography-tandem mass spectrometry (RP-LC-MS/MS) for proteomic analysis of LCM-acquired cells. This method was then applied to a clinical sample set consisting of basal, Her2-overexpressing and luminal A frozen tumor tissues as determined by prior microarray studies. Three tumors per group were dissected in triplicate, for a total of 27 samples. Each dissection consisted of approximately 10,000 cells, corresponding to 3-5 μg protein. Following LCM, the thermoplastic membranes containing the captured cells were peeled from the cap, suspended in SDS loading buffer and gently heated for 10 minutes. The solubilized proteins were electrophoresed 2 cm into a 10-20% Tricine gel followed by in-gel trypsin digestion and peptide extraction. Replicate LC-MS/MS analysis of the tryptic peptides from each of the 27 samples was performed on a Thermo LTQ XL ion trap mass spectrometer. The resultant tandem mass spectra were searched against the human IPI database using the Myrimatch search algorithm and the results filtered using IDPicker. A spectral counting approach was applied to the data-dependent data to compare quantitative differences among the identified proteins. A total of 91,646 spectra were confidently identified, corresponding to 1671 protein groups across all biological and technical replicates. An average of 545, 543, and 610 protein groups were identified in the basal, ERBB2, and luminal A subtypes, respectively, from an equivalent of approximately 800 cells. Spectral count differences between datasets were analyzed using both a Quasi-likelihood Poisson regression model, developed in-house, and the Limma package in Bioconductor. Using a False Discovery Rate < 0.05, a total of 90 proteins showed statistically significant differences in expression among the breast tumor subtypes. Hierarchical cluster analysis generated a heat map of the protein expression patterns. Novel proteins as well as proteins corresponding to genes previously shown by gene array studies to be specific to each tumor type, including NES, ERBB2 and LGALS3BP, were represented among the differentially expressed proteins. These results demonstrate the feasability of this method to identify differential expression of protein biomarkers among breast cancer subtypes. These biomarkers should yield new insights into the pathogenesis of breast cancer and could be rapidly adapted into clinical diagnostics to guide therapeutic decisions for individual patients. The possible role in breast cancer for the novel proteins identified is currently under investigation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-11.

Tibial Dyschondroplasia–Associated Proteomic Changes in Chicken Growth Plate Cartilage

Tibial dyschondroplasia (TD) is a poultry leg problem that affects the proximal growth plate of the tibia, preventing its transition to bone. To understand the disease-induced proteomic changes, we compared the protein extracts of cartilage from normal and TD-affected growth plates. TD was induced by feeding thiram to chickens 2 wk before tissue harvest. Proteins were extracted from whole tissues and from conditioned media (CM) prepared by incubating appropriate growth plate tissues in serum-free culture medium for 48 hr. The extracts were prefractionated to contain proteins ranging between 10 and 100 kD. Equal amounts of proteins were subjected to 2D gel electrophoresis with three individual samples per group. The gels were silver stained, and digital images were compared and analyzed with Melanie software to determine differentially expressed protein spots. On comparison of two sets of gels, 47 matching spots were detected in tissue extracts and 27 in CM extracts. Among the matching spots, 12 were determined to be down-regulated in tissue extracts (P < or = 0.05) and two in CM extracts (P < or = 0.05) of TD-affected growth plates. Altogether, 32 protein spots could be identified in both tissue and CM extracts by in-gel trypsin digestion, followed by peptide mass fingerprinting and mass spectrometry (MS)/MS fragmentation. The down-regulated proteins included alpha-enolase, G protein, origin recognition complex, peptidyl prolyl isomerase, calumenin, type II collagen precursor, and the expressed sequence tag pgm2n.pk014.f20, a protein with homology to human reticulocalbin-3 (RCN3). Most of the downregulated proteins are associated with signal transduction, energy metabolism, and secretory functions that are integral to cell viability. Consistent with our earlier findings that the TD chondrocytes are nonviable, the current results suggest that thiram very likely interferes with basic metabolic functions of chondrocytes, leading to their death and, consequently, to the pathogenesis of TD.

C-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.

Low-dose irradiation causes rapid alterations to the proteome of the human endothelial cell line EA.hy926

High doses of ionising radiation damage the heart by an as yet unknown mechanism. A concern for radiological protection is the recent epidemiological data indicating that doses as low as 100-500 mGy may induce cardiac damage. The aim of this study was to identify potential molecular targets and/or mechanisms involved in the pathogenesis of low-dose radiation-induced cardiovascular disease. The vascular endothelium plays a pivotal role in the regulation of cardiac function and is therefore a potential target tissue. We report here that low-dose radiation induced rapid and time-dependent changes in the cytoplasmic proteome of the human endothelial cell line EA.hy926. The proteomes were investigated at 4 and 24 h after irradiation at two different dose rates (Co-60 gamma ray total dose 200 mGy; 20 mGy/min and 190 mGy/min) using 2D-DIGE technology. Differentially expressed proteins were identified, after in-gel trypsin digestion, by MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. We identified 15 significantly differentially expressed proteins, of which 10 were up-regulated and 5 down-regulated, with more than ±1.5-fold difference compared with unexposed cells. Pathways influenced by the low-dose exposures included the Ran and RhoA pathways, fatty acid metabolism and stress response.

Unexpected presence of graminan- and levan-type fructans in the evergreen frost-hardy eudicot Pachysandra terminalis (Buxaceae): purification, cloning, and functional analysis of a 6-SST/6-SFT enzyme.

About 15% of flowering plants accumulate fructans. Inulin-type fructans with β(2,1) fructosyl linkages typically accumulate in the core eudicot families (e.g. Asteraceae), while levan-type fructans with β(2,6) linkages and branched, graminan-type fructans with mixed linkages predominate in monocot families. Here, we describe the unexpected finding that graminan- and levan-type fructans, as typically occurring in wheat (Triticum aestivum) and barley (Hordeum vulgare), also accumulate in Pachysandra terminalis, an evergreen, frost-hardy basal eudicot species. Part of the complex graminan- and levan-type fructans as accumulating in vivo can be produced in vitro by a sucrose:fructan 6-fructosyltransferase (6-SFT) enzyme with inherent sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan 6-exohydrolase side activities. This enzyme produces a series of cereal-like graminan- and levan-type fructans from sucrose as a single substrate. The 6-SST/6-SFT enzyme was fully purified by classic column chromatography. In-gel trypsin digestion led to reverse transcription-polymerase chain reaction-based cDNA cloning. The functionality of the 6-SST/6-SFT cDNA was demonstrated after heterologous expression in Pichia pastoris. Both the recombinant and native enzymes showed rather similar substrate specificity characteristics, including peculiar temperature-dependent inherent 1-SST and fructan 6-exohydrolase side activities. The finding that cereal-type fructans accumulate in a basal eudicot species further confirms the polyphyletic origin of fructan biosynthesis in nature. Our data suggest that the fructan syndrome in P. terminalis can be considered as a recent evolutionary event. Putative connections between abiotic stress and fructans are discussed.

Global and targeted proteomics in developing jack bean (Canavalia ensiformis) seedlings: an investigation of urease isoforms mobilization in early stages of development

Jack bean (Canavalia ensiformis) seeds are toxic for insects and the toxicity is due in part to an entomotoxic peptide enzymatically released from ureases in the midgut of susceptible insects. To characterize expression of urease isoforms in jack bean seed, particularly the more abundant urease isoform (JBU), quantitative proteomics was performed. Quiescent through 5-day germinating seeds were analyzed at 1-day intervals using a total proteomics approach (TPA) and also after co-immunoprecipitation (co-IP) with anti-JBU monoclonal antibodies. Jack bean proteins for TPA and co-IP were pre-fractionated by SDS-PAGE, segmented for in-gel trypsin digestion, and analyzed by liquid chromatography coupled to nanospray ionization tandem mass spectrometry (LC-MS/MS). Acquired MS(2) data were searched against a comprehensive plant database and the MEROPS peptidase database, in the absence of a jack bean EST database. Proteins detected in TPA were quantified by label-free spectral counting. A total of 234 and 106 non-redundant proteins were detected in TPA and co-IP, respectively. Mobilization of JBU was observed beginning 3-days after imbibition indicating that the entomotoxic peptide was not formed before this stage. A predicted urease isoform, JBURE-IIb, was detected in the co-IP study. Additionally, 46 plastid proteins, including RuBisCO and plastid ATPase were pulled down with JBU antibodies. These data shed new light on the behavior of urease isoforms during the early stages of plant development.

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI–TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as number of peaks detected, number of peptides identified, and sequence coverage, and the digestion time was reduced to 45 min. The gel/mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.

Abstract A68: Assessing dose response to kinase inhibitors using quantitative mass spectrometry

Abstract Goal: The goal of this experiment is to understand how kinase inhibitor therapies, like Erlotinib targeting the epidermal growth factor receptor (EGFR), modulate the signaling network using phosphoproteomics; developing a straightforward process to enable physicians to gather information that may assist in establishing a molecular basis for selection of chemotherapy, particularly small tyrosine inhibitor therapies, and for tracking patients’ responses to treatment. Methods: Non-small cell lung cancer (NSCLC) cell lines were used as model systems for examining lung cancer signaling, particularly the HCC827 cell line that overexpresses EGFR. To examine the response to inhibitor, EGFR from HCC827 cells was enriched by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation. After cell lysis, protein assays were carried out so that an equivalent amount of total protein was loaded into each lane. After excision of the appropriate region of the gel, reduction, and alkylation, in-gel trypsin digestion was performed. The resulting peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on a hybrid linear ion trap-orbital ion trap mass spectrometer. The high resolution and accurate mass measurements of the peptides enable relative quantification that can be used to examine dose response for each phosphorylation site. After detection and relative quantification of peptides including phosphorylation sites, quantitative assays based on liquid chromatography coupled to multiple reaction monitoring (LC-MRM) can be developed to increase sensitivity for subsequent translation to patient samples. Results: Using high resolution mass spectrometry, more than ten phosphotyrosine sites could be detected and quantified from EGFR and several of these phosphorylations were shown to be dose dependent to Erlotinib, a kinase inhibitor specific for EGFR. The decreases in phosphorylation at sites including tyrosine 1197 can be used as surrogates to monitor the inhibition of EGFR activity for determination of patient response. LC-MRM assays have been developed for EGFR expression and modification and implemented in the HCC827 cell line. Conclusion: The ability to monitor the effectiveness of the drug treatment at a molecular level allows physicians to modulate drug treatment based on the responses observed for each patient. This knowledge can maximize treatment effectiveness or suggest when other treatments may be more effective. The phosphoproteomics techniques illustrated here for the well characterized EGFR molecule can also be utilized to examine the drug induced molecular changes in other protein targets of clinical significance. Because of the potential importance of this experiment in clinical applications, a manuscript is being prepared for a chemical education journal that describes the lab activities and provides a virtual learning environment to monitor drug response with mass spectrometry. ∗This presenting student is supported by an ARRA supplement to the NCI Cancer Center Support Grant awarded to Moffitt (3P30 CA076292-11S6 PI WS Dalton) to create a training program for underrepresented undergraduate students in clinical proteomics Project LINK (Leaders In New Knowledge-Emerging Technologies) that also emphasizes education in health disparities and community outreach. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A68.

Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT). Following the reaction with the ICAT reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Based on the changes in proteins, we speculate modulation of cardiac muscle protein expression represents a fundamental alteration induced by chronic alcohol consumption, consistent with changes in myocardial wall thickness measured under the same conditions.

An antioxidant protein in Curcuma comosa Roxb. Rhizomes

Curcuma comosa Roxb. is an indigenous Thai herb which is usually used as a food ingredient but it is also used in traditional folk medicine for the treatment of uterine inflammation. The crude protein extract from the rhizomes of this plant was found to possess free radical scavenging capacity, as detected by the DPPH assay. This antioxidant activity was purified by DEAE anion exchange chromatography to a fraction (called IE-1) that was comprised of a single main protein band of ∼18 kDa (UB-DEAE), as determined by SDS–PAGE resolution with Coomassie blue staining, and had a specific activity of 193.8 U/mg. In-gel trypsin digestion of the SDS–PAGE resolved UB-DEAE band followed by liquid chromatography–tandem mass spectrometry produced three reliably sequenced peptides, which all were found to very likely be fragments of a superoxide dismutase homologue (SOD, EC 1.15.1.1), an antioxidant enzyme that has been found in several plants. In support of this notion, the IE-1 fraction was found to yield positive results with the riboflavin–nitroblue tetrazolium (NBT) assay, a standard test for SOD-like activity. Together, these data then support the existence of an SOD homologue antioxidant protein in the rhizomes of C. comosa as a contributing agent to the total observed antioxidant activity.

Conformation of Dimeric Apolipoprotein A-I Milano on Recombinant Lipoprotein Particles

Apolipoprotein A-I Milano (apoA-I(Milano)) is a naturally occurring human mutation of wild-type apolipoprotein A-I (apoA-I(WT)) having cystine substituted for arginine(173). Two molecules of apo-I(WT) form disks with phospholipid having a defined relationship between the apoA-I(WT) molecules. ApoA-I(Milano) forms cystine homodimers that would not allow the protein to adopt the conformation reported for apoA-I(WT). The conformational constraints for dimeric apoA-I(Milano) recombinant high-density lipoprotein (rHDL) disks made with phospholipid were deduced from a combination of chemical cross-linking and mass spectrometry. Lysine-selective homobifunctional cross-linkers were reacted with homogeneous rHDL having diameters of 78 and 125 A. After reduction, cross-linked apoA-I(Milano) was separated from monomeric apoprotein by gel electrophoresis and then subjected to in-gel trypsin digest. Cross-linked peptides were confirmed by MS/MS sequencing. The cross-links provided distance constraints that were used to refine models of lipid-bound dimeric apoA-I(Milano). These studies suggest that a single dimeric apoA-I(Milano) on 78 A diameter rHDL girdles the edge of a phospholipid disk assuming a "belt" conformation similar to the "belt" region of apoA-I(WT) on rHDL. However, the C-terminal end of dimeric apoA-I(Milano) wraps around the periphery of the particle to shield the fatty acid chains from water rather than folding back onto the "belt" as does apoA-I(WT). The two apoA-I(Milano) dimers on a 125 A diameter rHDL do not encircle the periphery of a phospholipid disk but appear to reside on the surface of a laminar micelle.

Abstract 3746: Proteomic analysis of laser capture microdissected cells from basal, Her 2 positive and luminal A human breast tumors

Abstract The analysis of tissue is often confounded by the heterogenous nature, small size of biopsies, and focal presence of tumors. The use of laser capture microdissection (LCM) allows for isolation of nearly pure cell populations enhancing specificity of subsequent analyses. A method developed in our laboratory facilitating the removal of adhered cells utilizing SDS-PAGE followed by in-gel digestion and tandem mass spectrometry was applied to LCM-acquired cells from Basal, Her 2 positive and Luminal A breast tumor tissues. Cells were LCM-acquired from frozen tissue sections corresponding to Basal, Her 2 positive or Luminal A human breast tumor tissue. The sample set consisted of 3 tumors per subgroup with 3 caps per tumor, for a total of 27 caps each containing approximately 10,000 cells corresponding to 3-5 μg protein. The thermoplastic membranes containing the captured cells were peeled from the cap and suspended in SDS loading buffer and gently heated for 10 minutes. The solubilized proteins were electrophoresed 2 cm into a 10-20% Tricine gel followed by in-gel trypsin digestion and peptide extraction. Tryptic peptides were analyzed by reversed-phase (RP) liquid chromatography-tandem mass spectrometry (LC-MS-MS) on a Thermo Orbitrap instrument. Following LC/MS/MS analysis of the LCM digests, tandem spectra were searched against the human IPI database using the search algorithm, Myrimatch, and the search results filtered using IDPicker. A spectral counting approach was applied to the data-dependent data to compare quantitative differences of proteins identified. A total of 91646 spectra were confidently identified which corresponded to 1671 protein groups across all biological and technical replicates. An average of 545, 543, and 610 protein groups were identified in the basal, Her 2 positive, and Luminal A subtypes, respectively, from an equivalent of roughly 800 cells. Spectral count differences between datasets between normal and tumor samples were analyzed using both a Quasi-likelihood Poisson regression model, developed in-house, in addition to using the Limma package in Bioconductor. Using the criteria of a False Discovery Rate (FDR) &amp;lt; 0.05, a total of 90 proteins showed statistically significant differences in expression among the breast tumor subtypes. A hierarchical clustering of these proteins was performed, creating a heat map representing the gene expression patterns of these proteins. As expected, ERBB2 was identified in the Her 2 positive tumor tissue samples; however, this protein was not identified in either the Basal or Luminal A samples. The proteins identified which were found to distinguish each tissue subtype are currently being investigated for their biological relevance in breast cancer pathways and as possible prognostic and predictive biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3746.

Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

BackgroundPoly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions.ResultsPARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1.ConclusionsThis study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism.

PTU-083 Proteomic analysis of bile in benign and malignant pancreatobiliary disease

<h3>Introduction</h3> Obstruction of the biliary tree has a variety of aetiologies. The correct diagnosis is crucial for appropriate definitive therapy. A major differential diagnosis of biliary obstruction is pancreatic adenocarcinoma (PA). PA is the 10th most common cancer in the UK with mortality similar to incidence. Diagnosis of PA relies on a combination of cross-sectional imaging and serum biomarkers, but only a minority of cases are diagnosed correctly and early enough for curative resection. Earlier diagnostic certainty would be likely to improve outcome and guide therapeutic strategy. Proteomic profiling is a promising technique for new biomarker discovery, but has rarely been successfully carried out in human bile. This project (1) analyses and compares the proteomes of bile from PA patients vs. bile from patients with benign biliary disease and (2) seeks to identify potential protein biomarkers that can differentiate PA from benign biliary disease. <h3>Methods</h3> Bile was aspirated at ERCP from patients with benign or malignant biliary obstruction. Particulate matter was removed by centrifugation and equal volumes subjected to SDS-PAGE. In-gel trypsin digestion was performed and the resulting peptides analysed by tandem mass spectrometry. Proteins were identified using SEQUEST to search the human RefSeq protein sequence database. Progenesis software was used to compare relative levels of proteins between patient groups. <h3>Results</h3> Proteomic analysis of bile from four patients with PA and four with benign biliary obstruction was performed. Over 100 different proteins were identified, including pancreatic gastric triacylglycerol lipase, carboxypeptidase B and bile salt-activated lipase, confirming that the profile of proteins represents the biliary proteome. Several differentially expressed proteins were identified including upregulation of carcinoembryonic antigen–related cell adhesion molecule 6 (CEACAM6) in the malignant group. <h3>Conclusion</h3> This work confirms that bile is amenable to proteomic analysis and that differences in the biliary proteome can be detected in malignant and non-malignant cases. We confirmed earlier reports of CEACAM6 overexpression in PA. We are currently analysing the data further to identify additional candidate biomarker proteins. Future work will extend this approach to the study of bile in other diseases, such as cholangiocarcinoma.

Direct Ubiquitination of β-Catenin by Siah-1 and Regulation by the Exchange Factor TBL1

Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.

Identification of molecular markers for pre-engraftment immune reactions after cord blood transplantation by SELDI-TOF MS

Cord blood transplantation (CBT) is frequently associated with pre-engraftment immune reaction (PIR), which is characterized by high-grade fever that peaks around day 9 of transplantation. PIR mimics hyperacute GVHD or engraftment syndrome; however, it is considered to be of different etiology as it occurs before engraftment. Proteomic patterns have been studied in the fields of transplantation, but no specific marker has been identified. As there are no data to confirm the mechanism of PIR, we used a surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF MS) system to identify a specific marker for PIR. The protein expression profile of serum samples from CBT patients was analyzed with a SELDI-TOF MS system. A protein peak that commonly predominated in PIR was purified by an anion exchange column, isolated by SDS-PAGE, and identified by in-gel trypsin digestion, and mass fingerprinting. A 8.6-kDa protein and 11-kDa protein that increased by 10- to 100-fold in the serum of patients during PIR was identified as anaphylatoxin C4a and serum amyloid A. SELDI-TOF MS system in combination with other proteomic methods could serve as a potential diagnostic tool in discovering biomarkers for PIR after CBT.

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and alpha-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and alpha-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric alpha-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of alpha-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.

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PROTEOMICS – Clinical ApplicationsVolume 4, Issue 1 In this issue In this issue First published: 13 January 2010 https://doi.org/10.1002/prca.201090002AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Renal rejection: your kidney don't love me no more pp. 32–47 Sometimes getting a new kidney installed is the easy part – it's getting it to stay there and not feel rejected by its new host that is the hard part. That and not coming down with some nasty disease due to enhanced levels of immunosuppressive therapy while trying to avoid rejection. That and suffering from the biopsy procedure when other things don't work. Transplants are no joke. Sigdel et al. took the shotgun approach to the problem, looking for urinary proteomic markers for rejection. Using a semi-quantitative shotgun meant no labeled species, just counting the fragments of the desired size as they whizzed by. Three proteins have been further validated by ELISA performance. Likely candidates are UMOD, SERPINF1 and CD44. Adenoma: unique pattern midway to carcinoma pp. 60–70 It's a thoughtful cancer that gives the body a reasonable warning before it begins a frontal assault. But that, apparently, is what colorectal carcinoma (CRC) does. The early-stage CRC begins life as an adenoma, which can be treated prospectively, before progressing. For those too young to have been subjected to the ignominy of a colonoscopic exam, a simple swab test would be greatly appreciated. Surely with such a kindness in mind, Lam et al. embarked on a 2-D electrophoretic hunt for markers that could distinguish normal from adenoma tissue. Working with 20 paired sets of normal and adenoma tissue samples, they identified over 1000 proteins by 2-DE and MALDI-TOF/TOF. They found four proteins up-regulated (Annexin A3, S100A11, eIF5A-1, S100P) and three down-regulated (S100A9, Galectin-1, and FABPL) by at least twofold in both cases. Functionally, they made sense. Now for a business plan… Not so mighty mouse: plasma proteomics of mouse model of Duchenne Muscular Dystrophy pp. 71–83 The most common way to study Duchenne Muscular Dystrophy (DMD) is to work with a mouse model of the disease – it is convenient but current markers are not reliably applicable to larger animals. Colussi et al. needed markers for their studies of histone deacetylase inhibitors, drugs which restore some muscular strength in mdx (mutant) mice. In this article, they report their proteomic analysis of the effects of the drug SAHA on plasma protein levels. After establishing baseline levels for wt versus mdx, they then looked for differences between mdx and mdx+SAHA. At least eight differences were detected in wt versus mdx, three up- and five down-regulated. For mdx versus mdx+SAHA, at least four changes were observed, including normalization of two proteins. Analysis was done by 2-DE, in-gel trypsin digestion and LC-MS/MS. Volume4, Issue1January 2010 RelatedInformation