Membrane-enriched Fraction Research Articles

In this issue

PROTEOMICS – Clinical ApplicationsVolume 4, Issue 5 In this issue In this issue First published: 29 April 2010 https://doi.org/10.1002/prca.201090018AboutPDF 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 When BRCA1 breaks pp. 489–498 The most frequent cancer among women, breast cancer arises principally due to mutations in the tumor suppressor genes, BRCA1 and BRCA2/FANCD1. Carriers of BRCA1 mutations have a 50–80% chance of breast cancer by age 70. BRCA1 is highly multi-functional, its most critical function being repair of double-strand breaks in DNA before replication. Other jobs include protein degradation, cell cycle control, transcription regulation, and apoptosis control. A major question is which (if any) of these or other factors are involved in the progression of BRCA1 tumors. Bouley et al. approached the problem using isogenic (+/− Knockdown) strains of HeLa cells, liquid phase IEF and DIGE first and second dimensions, then MALDI-TOF MS to identify proteins significantly shifted in amount. Particularly interesting was the over-expression of replication protein A 1 (RPA1), and RPA2 in HeLaKD cells. Deconstructing the barrier pp. 538–549 Understanding the normal functioning of the human placenta during the course of pregnancy is critical for dealing with the diseases that endanger both mother and fetus. Communication between the two parties occurs primarily through the apical plasma membrane of the syncytiotrophoblast (STB). Here occur exchange of nutrients and gas, transfer of hormones and growth factors, removal of waste, and immunological processing. The membrane is polarized, with the microvillous side in direct contact with maternal blood. Zhang et al. prepared vesicles from the highly enriched membrane fraction, which were analyzed by nano-LC ESI-MS/MS. A total of 296 proteins were identified, some assigned to the placenta for the first time. They found raft domains, markers and anchors; calcium transporters, and signal transduction; a new view of the healthy placenta for future reference. Osteosarcoma: spots tell the tale early pp. 560–567 Primary osteosarcoma (OS) is one of childhood's most common malignant bone tumors. As a bit of cruel humor, it is separable into two forms — a good responder and a poor responder to chemotherapeutic treatment — after chemo. Surgery plus chemo gave cure rates in the vicinity of 70%, but there were some choices in pretreatment that were best made with foreknowledge of responder type. Kikuta et al. were aware of hints of biomarkers found by others, so they elected to explore with 2-D DIGE. Out of 2250 protein spots, they found 39 that differed depending on the responder status. One of the best markers was peroxiredoxin 2 (PRDX 2), even after the validation by a Western blot on an additional set of four patient samples. It looks promising. Volume4, Issue5May 2010 RelatedInformation

Differential regulation of ventral spinal atypical PKC and ERK MAP kinase following hypocapnia‐induced respiratory neural inactivity

Hypocapnia‐induced respiratory neural inactivity elicits mechanisms of plasticity that enhance phrenic motor output once activity is restored, an effect known as inactivity‐induced phrenic motor facilitation (iPMF). Since atypical protein kinase C (aPKC) activity is necessary for iPMF, we tested the hypothesis that iPMF is associated with increased aPKC expression in spinal regions associated with the phrenic motor nucleus. Anesthetized and ventilated rats were exposed to 30 min of hypocapnia (5–10 mmHg below baseline) to reversibly stop respiratory neural activity. Following hypocapnia, ventral C3–C5 was fractionated into membrane‐enriched, cytoplasm‐enriched and nucleus‐enriched fractions. Total and phosphorylated aPKC increased in membrane‐enriched (47 +/− 10 and 39 +/− 11%, respectively, p<0.05), but decreased in cytoplasm‐enriched fractions (−77 +/− 5 and −74 +/− 6%, respectively, p<0.05) post‐hypocapnia. In contrast, there were no significant changes in membrane or cytoplasmic expression of phosphorylated ERK (−41 +/− 14 and −12 +/− 10%, respectively, p>0.05), a kinase implicated in other forms of spinal plasticity. Phospho‐ERK was increased in nuclear fractions post‐hypocapnia (97 +/− 17%, p<0.05), suggesting a possible role for ERK in longer‐lasting forms of plasticity elicited by neural inactivity. These data support a prominant role for aPKC in iPMF following hypocapnia (NIH HL69064).

Identification and Characterization of TwoBordetella aviumGene Products Required for Hemagglutination

Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

Differential expression of pathogen-recognition molecules between dendritic cell subsets revealed by plasma membrane proteomic analysis

Dendritic cells (DC) are comprised of several subsets with distinct functions, differing in their capacity to respond to pathogen products. To gain novel insights into their pathogen specificity, we compared the protein composition of the plasma membrane of CD8+ and CD8− DC, directly isolated from mouse spleens. Differences in protein expression were determined using semi-quantitative high-resolution mass spectrometry of label-free plasma membrane-enriched fractions. Our comparative proteomic analysis detected over 1500 proteins, revealing broad differences in expression of pathogen receptors, adhesion molecules and T-cell regulatory molecules. Many of these findings were validated using flow cytometry and Western Blot analysis of integral and luminal surface-associated membrane proteins. This analysis provides major advantages over genomic approaches as it directly measures protein expression at a particular location. Our study highlights the diversity of surface protein expression amongst components of the DC network.

Synaptic and Endosomal Localization of Active γ-Secretase in Rat Brain

BackgroundA key player in the development of Alzheimer's disease (AD) is the γ-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. γ-Secretase is crucial for the generation of the neurotoxic amyloid β-peptide (Aβ) but also takes part in the processing of many other substrates. In cell lines, active γ-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active γ-secretase in the affected organ of AD, namely the brain.Principal FindingsWe show by subcellular fractionation of rat brain that high γ-secretase activity, as assessed by production of Aβ40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Aβ40 production and contained high amounts of the γ-secretase components. Further purification of the synaptic vesicles verified the presence of the γ-secretase components in these compartments. The localization of an active γ-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated γ-secretase inhibitor together with confocal microscopy.SignificanceThe information about the subcellular localization of γ-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active γ-secretase.

Immunoenzymatic assay for the diagnosis of American tegumentary leishmaniasis using soluble and membrane‐enriched fractions from infectious Leishmania (Viannia) braziliensis

The diagnosis of American tegumentary leishmaniasis (ATL) is based on the visualization or isolation of the parasite, which is a time-consuming and poorly sensitive method. In this study, we evaluated the accuracy and reliability of ELISA for the diagnosis of ATL using soluble (SF) and membrane-enriched (MF) antigen fractions obtained from an infectious strain of Leishmania (Viannia) braziliensis. A total of 152 serum samples investigated at a referral center in Rio de Janeiro, Brazil, between 2005 and 2007 were studied. Each sample was tested twice with each fraction for the calculation of reliability (intraclass coefficient (ICC)). Cut-off values of 0.22 (SF) and 0.33 (MF) were defined. The use of the fractions resulted in good discrimination between patients, with a large area under the curve (P<0.0001), but no difference was observed between the two fractions (P=0.45). Sensitivity was 89.5% for each fraction, specificity was 89.5% for SF and 93.4% for MF, and the positive likelihood ratio was 8.5 for SF and 13.6 for MF. The ICCs were excellent (SF: 0.96 and MF: 0.90). The antigens tested provided precision and accuracy for the diagnosis of ATL, with SF being recommended due to its lower cost and greater practicality.

Identification of Salt-Tolerant Sinorhizobium sp. Strain BL3 Membrane Proteins Based on Proteomics

Sinorhizobium sp. BL3 is a salt-tolerant strain that can fix atmospheric nitrogen in symbiosis with leguminous host plants under salt-stress conditions. Since cell membranes are the first barrier to environmental change, it is interesting to explore the membrane proteins within this protective barrier under salt stress. The protein contents of membrane-enriched fractions obtained from BL3 were analyzed by nanoflow liquid chromatography interfaced with electrospray ionization tandem mass spectrometry. A total of 105 membrane proteins were identified. These proteins could be classified into 17 functional categories, the two biggest of which were energy production and conversion, and proteins not in clusters of orthologous groups (COGs). In addition, a comparative analysis of membrane proteins between salt-stressed and non-stressed BL3 cells was conducted using a membrane enrichment method and off-line SCX fractionation coupled to nanoLC-MS/MS. These techniques would be useful for further comparative analysis of membrane proteins that function in the response to environmental stress.

Transient Upregulation of Protein Kinase C in Pressure-Overloaded Neonatal Rat Myocardium

Protein kinase C (PKC) appears to play a significant role in the signal transduction of cardiac growth and development. The aim of this study was to determine changes in the total PKC activity and the expression of PKC isoforms alpha, delta and epsilon in the rat heart that was affected by pressure overload imposed at postnatal day (d) 2. Three groups of Wistar rats were employed for the experiment: rats submitted to the abdominal aortic constriction (AC), sham-operated controls (SO) and intact controls. Animals were sacrificed at d2, d3, d5 and d10. The total PKC activity was measured by the incorporation of (32)P into histone IIIS and the expression of PKC was analyzed by immunoblotting in the homogenate of the left ventricular myocardium and in the cytosolic, membrane-enriched (10(5) g) and nuclear-cytoskeletal-myofilament-enriched (10(3) g) fractions. We observed the significant transient increase in both the total PKC activity and the expression of all isoforms at d5 (the third day after the operation) in the cardiac homogenate of AC rats as compared with SO animals. Aortic constriction did not significantly affect the distribution of activity and isoform abundance among individual cellular fractions except for PKCdelta, which increased significantly at d10 in the cytosolic fraction at the expense of the membrane-enriched fraction. It is concluded that PKCalpha, PKCdelta and PKCepsilon undergo transient upregulation associated with the accelerated cardiac growth induced by pressure overload imposed in the very early postnatal period.

Long-term vitamin A supplementation at therapeutic doses induces mitochondrial electrons transfer chain (METC) impairment and increased mitochondrial membrane-enriched fraction (MMEF) 3-nitrotyrosine on rat heart

The aim of the present study was to compare electrons flux and oxidative/nitrosative stress parameters on the heart among rats supplemented with vitamin A and one not supplemented long-term. Vitamin A has important roles for the cardiovascular system as well as antioxidant properties. However, pro-oxidant properties have been reported. Male adult rats were treated with four different doses of retinyl palmitate (1000–9000 IU/Kg/day) or saline (control) for 28 days and the heart was removed for analysis. Electrons flux and oxidative/nitrosative stress parameters were evaluated and statistics were conducted with Anova one-way followed by Dunnet's post hoc and significance level of p<0.05. The supplementation induced increase on lipids/proteins oxidation and mitochondrial 3-nitrotyrosine content, an imbalance on enzymatic activity and a decrease on respiratory chain complexes activities. The results suggest that vitamin A induces oxidative/nitrosative stress and mitochondrial impairment on a cardiac level.

Abstract B63: Apaf-1 mislocalization is a strong indicator of drug resistance in B cell lymphomas

Abstract Resistance to chemotherapy remains a challenge in the clinical management of diffuse B cell lymphomas despite aggressive chemotherapy such as CHOP and monoclonal CD20. We previously reported that sequestration of Apaf-1 to membrane lipid rafts was responsible for apoptosis resistance in B-cell lymphoma cell lines1. Here, we extended our studies to 60 clinical biopsies from patients with B-cell lymphomas, T-cell lymphomas and reactive lymphadenopathy, to investigate if the resistance to drug-induced apoptosis was, indeed, a function of Apaf-1 mislocalization. Firstly, cells were separated from these biopsies and their sensitivity to a variety of apoptosis inducing agents was assessed. Whereas, most T cell lymphomas as well as reactive lymphadenopathy cells were sensitive to apoptotic stimuli, B cell lymphomas exhibited strong resistance. We then investigated the expression of Apaf-1 and its intracellular localization in these clinical biopsies. To do so, cell fraction was performed to separate cytosol from membrane-enriched fractions. The latter were further subjected to density gradient centrifugation to obtain lipid raft fractions. We show that Apaf-1 was expressed in total cell lysates from B and T cell lymphomas, however upon fractionation the localization was strikingly different. In T cell lymphoma samples as well as in cells derived from reactive lymphadenopathy biopsies, Apaf-1 expression was prominently detected in the cytosol, which correlated with the sensitivity of the cells to apoptotic stimuli. In contrast, whereas cytosolic Apaf-1 expression was significantly lower or absent in almost all B cell lymphomas analyzed, increased localization of the protien was detected in membrane lipid rafts. The latter was confirmed by immunohistochemical analysis of tissues from the same biopsy specimens. Interestingly, the resistance of B cell lymphomas to apoptotic execution (drug-induced or death receptor-mediated) was significantly bypassed upon incubation of cells with pharmacological agents that facilitated the dissociation of Apaf-1 from the lipid rafts to the cytosol. Taken together, our results implicate Apaf-1 mislocalization as a potential diagnostic marker for B-cell lymphomas as well as a predictor of response to therapeutic management. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B63.

Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 microg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 microg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains.

Large-Scale Phosphoprotein Analysis inMedicago truncatulaRoots Provides Insight into in Vivo Kinase Activity in Legumes

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.

Inhibition of specific adenylyl cyclase isoforms by lithium and carbamazepine, but not valproate, may be related to their antidepressant effect

Lithium, valproate, and carbamazepine decrease stimulated brain cyclic-AMP (cAMP) levels. Adenylyl cyclase (AC), of which there are nine membrane-bound isoforms (AC1-AC9), catalyzes the formation of cAMP. We have recently demonstrated preferential inhibition of AC5 by lithium. We now sought to determine whether carbamazepine and valproate also preferentially inhibit specific AC isoforms or decrease cAMP levels via different mechanisms. COS7 cells were transfected with one of AC1-AC9, with or without D1-dopamine receptors. Carbamazepine's and valproate's effect on forskolin- or D1 agonist-stimulated ACs was studied. The effect of Mg(2+) on lithium's inhibition was studied in membrane-enriched fraction from COS7 cells co-expressing AC5 and D1 receptors. AC5 knockout mice were tested for a behavioral phenotype similar to that of lithium treatment. Carbamazepine preferentially inhibited forskolin-stimulated AC5 and AC1 and all D1 agonist-stimulated ACs, with AC5 and AC7 being the most sensitive. When compared to 1 or 3 mM Mg(2+), 10 mM Mg(2+) reduced lithium-induced AC5 inhibition by 70%. In silico modeling suggests that among AC isoforms carbamazepine preferentially affects AC1 and AC5 by interacting with the catechol-estrogen site. Valproate did not affect any forskolin- or D1 receptor-stimulated AC. AC5 knockout mice responded similarly to antidepressant- or lithium-treated wild-types in the forced-swim test but not in the amphetamine-induced hyperactivity mania model. Lithium and carbamazepine preferentially inhibit AC5, albeit via different mechanisms. Lithium competes with Mg(2+), which is essential for AC activity; carbamazepine competes for AC's catechol-estrogen site. Antidepressant-like behavior of AC5 knockout mice in the forced-swim test supports the notion that AC5 inhibition is involved in the antidepressant effect of lithium and carbamazepine. The effect of lithium and carbamazepine to lower cAMP formation in AC5-rich dopaminergic brain regions suggests that D1-dopamine receptors in these regions are involved in the antidepressant effect of mood stabilizers.

Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

Protein alterations induced by long‐term agonist treatment of HEK293 cells expressing thyrotropin‐releasing hormone receptor and G11α protein

This study aimed to determine whether sustained stimulation with thyrotropin-releasing hormone (TRH), a peptide with important physiological functions, can possibly affect expression of plasma membrane proteins in HEK293 cells expressing high levels of TRH receptor and G(11)alpha protein. Our previous experiments using silver-stained two-dimensional polyacrylamide gel electrophoretograms did not reveal any significant changes in an overall composition of membrane microdomain proteins after long-term treatment with TRH of these cells (Matousek et al. 2005 Cell Biochem Biophys 42: 21-40). Here we used a purified plasma membrane fraction prepared by Percoll gradient centrifugation and proteins resolved by 2D electrophoresis were stained with SYPRO Ruby gel stain. The high enrichment in plasma membrane proteins of this preparation was confirmed by a multifold increase in the number of TRH receptors and agonist stimulated G-protein activity, compared to postnuclear supernatant. By a combination of these approaches we were able to determine a number of clearly discernible protein changes in the plasma membrane-enriched fraction isolated from cells treated with TRH (1 x 10(-5) M, 16 h): 4 proteins disappeared, the level of 18 proteins decreased and the level of 39 proteins increased. Our concomitant immunochemical determinations also indicated a clear down-regulation of G(q/11)alpha proteins in preparations from hormone-treated cells. In parallel, we observed decrease in caspase 3 and alterations in some other apoptotic marker proteins, which were in line with the presumed antiapoptotic effect of TRH.

Improved Membrane Proteomics Coverage of Human Embryonic Stem Cells by Peptide IPG-IEF

Protein biomarkers are fundamental tools for the characterization of stem cells and for tracking their differentiation and maturation down developmental lineages. Technology development allowing increased coverage of difficult cellular proteomes should allow for the discovery of new and novel membrane protein biomarkers for use by the stem cell research community. The amphipathic and highly hydrophobic nature and relative low abundance of many membrane proteins present significant analytical challenges. These difficulties are amplified when the source material (tissue or cells) is only available in limited quantities (e.g., embryonic stem cells). Recent advances in enrichment for purer membrane fractions, the enzymatic and chemical digestion of membrane proteins in the presence of solvents or chaotropes, and the use of "shotgun" proteomics methodologies have gradually resulted in increased membrane proteome coverage with numbers of predicted integral membrane proteins now in excess of 1000 being routinely reported. We have recently demonstrated the advantages of using peptide isoelectric focusing in the first dimension on immobilized pH gradients (peptide IPG-IEF) followed by reversed phase chromatography and tandem MS to increase membrane proteome coverage. This study looked at achieving a similar level of membrane proteome coverage using modifications to reported methodologies while restricting the number of characterized human embryonic stem cells to 10(7) cells. Two-thousand two-hundred and ninety-two (2292) nonredundant proteins were identified with two or more high accuracy peptide matches from 260 mug of a human embryonic stem cell membrane enriched fraction with a false discovery rate of 0.32%. Gene Ontology (GO) mapping predicted 1279 (44.9%) of this list to be membrane proteins of which 395 proteins were predicted to be derived from the plasma membrane compartment. The TMHMM algorithm predicted 904 integral membrane proteins with up to 16 transmembrane helices. Collectively, we assert that the substantial membrane proteome coverage achieved using these procedures will enable rapid advances in the identification and quantitation of novel membrane proteins as markers of differentiation status and/or genetic mutation from relatively low numbers of cultured embryonic stem cells.

Comparative analysis of OFFGel, strong cation exchange with pH gradient, and RP at high pH for first‐dimensional separation of peptides from a membrane‐enriched protein fraction

The analysis and quantitation of membrane proteins have proved challenging for proteomics. Although several approaches have been introduced to complement gel-based analysis of intact proteins, the literature is rather limited in comparing major emerging approaches. Peptide fractionation using IEF (OFFGel), strong cation exchange HPLC using a pH gradient (SCX-pG), and RP HPLC at high pH, have been shown to increase peptide and protein identification over classic MudPIT approaches. This article compares these three approaches for first-dimensional separation of peptides using a detergent phase (Triton X-114) enriched membrane fraction from mouse cortical brain tissue. Results indicate that RP at high pH (pH 10) was superior for the identification of more peptides and proteins in comparison to the OFFGel or the SCX-pG approaches. In addition, gene ontology analysis (GOMiner) revealed that RP at high pH (pH 10) successfully identified an increased number of proteins with "membrane" ontology, further confirming its suitability for membrane protein analysis, in comparison to SCX-pG and OFFGel techniques.

ITRAQ-based Proteomics Profiling Reveals Increased Metabolic Activity and Cellular Cross-talk in Angiogenic Compared with Invasive Glioblastoma Phenotype

Malignant gliomas (glioblastoma multiforme) have a poor prognosis with an average patient survival under current treatment regimens ranging between 12 and 14 months. The tumors are characterized by rapid cell growth, extensive neovascularization, and diffuse cellular infiltration of normal brain structures. We have developed a human glioblastoma xenograft model in nude rats that is characterized by a highly infiltrative non-angiogenic phenotype. Upon serial transplantation this phenotype will develop into a highly angiogenic tumor. Thus, we have developed an animal model where we are able to establish two characteristic tumor phenotypes that define human glioblastoma (i.e. diffuse infiltration and high neovascularization). Here we aimed at identifying potential biomarkers expressed by the non-angiogenic and the angiogenic phenotypes and elucidating the molecular pathways involved in the switch from invasive to angiogenic growth. Focusing on membrane-associated proteins, we profiled protein expression during the progression from an invasive to an angiogenic phenotype by analyzing serially transplanted glioma xenografts in rats. Applying isobaric peptide tagging chemistry (iTRAQ) combined with two-dimensional LC and MALDI-TOF/TOF mass spectrometry, we were able to identify several thousand proteins in membrane-enriched fractions of which 1460 were extracted as quantifiable proteins (isoform- and species-specific and present in more than one sample). Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phenotype. The robustness of the data was corroborated by extensive bioinformatics analysis and by validation of selected proteins on tissue microarrays from xenograft and clinical gliomas. The data point to enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-angiogenic phenotype. In conclusion, we describe molecular profiles that reflect the change from an invasive to an angiogenic brain tumor phenotype. The identified proteins could be further exploited as biomarkers or therapeutic targets for malignant gliomas.

Peptide Separations by On-Line MudPIT Compared to Isoelectric Focusing in an Off-Gel Format: Application to a Membrane-Enriched Fraction from C2C12 Mouse Skeletal Muscle Cells

High-resolution peptide separation is pivotal for successful shotgun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3-10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides), while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88% of all peptides binned to a single fraction, as compared to 61% of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.

The Ubiquitin–Proteasome System Regulates the Stability of Neuronal Nicotinic Acetylcholine Receptors

Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin-proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the alpha3, beta2, and beta4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of alpha3 and the two beta subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the alpha7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.