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ABSTRACT: During mitosis, the Golgi membranes in mammalian cells undergo a continuous disassembly process and generate mitotic fragments that are distributed into the daughter cells and reassembled into new Golgi after mitosis. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins that are associated with interphase and mitotic Golgi membranes using quantitative proteomics that combines the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and LC-MALDI-MS/MS. A total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories: Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, and secretory and cytoskeletal proteins. More importantly, the combination of the quantitative proteomic approach with Western blot analysis allowed us to unveil 86 proteins with significant changes in abundance under the mitotic condition compared to the interphase condition. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that controls the Golgi disassembly and reassembly processes in the cell cycle.
Methods in molecular biology (Clifton, N.J.) 01/2012; 909:125-40.
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ABSTRACT: Tranche is a distributed repository designed to redundantly store and disseminate data sets for the proteomics community. It has several important features for researchers, including support for large data files, prepublication access controls, licensing options, and ensuring both data provenance and integrity. Tranche tightly integrates with ProteomeCommons.org, an online community resource that offers a variety of useful tools for proteomics researchers, including project management and data annotation. In this chapter, we discuss the development of Tranche and ProteomeCommons.org, paying particular attention to why it is desirable that data be publicly available and unrestricted as well as the challenges facing data archiving and open access. We then provide a technical overview of Tranche and ProteomeCommons.org as well as step-by-step instructions for using these resources, including the graphical user interface (GUI ), command-line tools, and Application Programmer Interface (API). We end with a brief discussion of current and future development efforts and collaborations.
Methods in molecular biology (Clifton, N.J.) 01/2011; 696:123-45.
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ABSTRACT: The endoplasmic reticulum (ER) is a multifunctional intracellular organelle responsible for the synthesis, processing and trafficking of a wide variety of proteins essential for cell growth and survival. Therefore, comprehensive characterization of the ER proteome is of great importance to the understanding of its functions and has been actively pursued in the past decade by scientists in the proteomics field. This review summarizes major proteomic studies published in the past decade that focused on the ER proteome. We evaluate the data sets obtained from two different organs, liver and pancreas each of which contains a primary cell type (hepatocyte and acinar cell) with specialized functions. We also discuss how the nature of the proteins uncovered is related to the methods of organelle purification, organelle purity and the techniques used for protein separation prior to MS. In addition, this review also puts emphasis on the biological insights gained from these studies regarding the molecular functions of the ER including protein synthesis and translocation, protein folding and quality control, ER-associated degradation and ER stress, ER export and membrane trafficking, calcium homeostasis and detoxification and drug metabolism.
Proteomics 11/2010; 10(22):4040-52. · 4.43 Impact Factor
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ABSTRACT: The fragmentation characteristics of peptides derivatized at the side-chain epsilon-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C(zeta)-N(epsilon) bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n - 1)(+)-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzyl-modified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary epsilon-amino group of the benzyl-derivatized lysyl side chain. If the secondary epsilon-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary epsilon-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.
Journal of the American Society for Mass Spectrometry 09/2010; 21(9):1624-32. · 4.00 Impact Factor
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ABSTRACT: During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, cargo, and cytoskeletal proteins. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil 84 proteins with significant changes in abundance under the mitotic condition compared with the interphase condition. Among these proteins, several COPI coatomer subunits (alpha, beta, gamma, and delta) are of particular interest. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.
Journal of Biological Chemistry 03/2010; 285(10):7197-207. · 4.77 Impact Factor
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ABSTRACT: Microparticles (MP) are submicron size membrane vesicles released from activated cells that are associated with thrombosis and inflammation. MP present diverse biological expressions that may be linked to a unique subset of proteins derived from their origin cells.
To identify these proteins, plasma samples were taken from 9 patients with deep venous thrombosis (DVT) documented by duplex ultrasound, 9 with leg pain but negative for DVT by duplex, and 6 healthy controls without a history of thrombosis, for fold variation. MP were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all human sequences. For protein identification, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein levels of DVT patients to baseline. The proteomic analysis was performed twice for each blood sample. Proteins were considered elevated or depressed if the iTRAQ ratio (R) deviated by 20% change from normal and a p-value less than 0.05.
Two proteins (Galectin-3 Binding Protein, [Gal3BP], R=1.76 and Alpha-2 macroglobulin [A2M] R=1.57) were differentially expressed on DVT patients. Nine proteins were depleted including fibrinogen beta and gamma chain precursors (R=0.65).
These proteins influence thrombosis through inflammation, cell shedding, inhibition of fibrinolysis and hemostatic plug formation. Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis in humans.
Thrombosis Research 02/2010; 125(6):e269-74. · 2.44 Impact Factor
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Venkateshwar G Keshamouni,
Pratik Jagtap,
George Michailidis,
John R Strahler,
Rork Kuick,
Ajaya Kumar Reka,
Panagiotis Papoulias,
Rashmi Krishnapuram,
Anjaiah Srirangam,
Theodore J Standiford, Philip C Andrews,
Gilbert S Omenn
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ABSTRACT: To gain insights into how TGF-beta regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, including newly associated proteins calpain, fascin and macrophage-migration inhibitory factor (MIF). Comparing protein and mRNA expression overtime showed that all the 14 up-regulated proteins involved in the actin-cytoskeleton remodeling were accompanied by increases in corresponding mRNA expression. Interestingly, siRNA mediated knockdown of cofilin1 potentiated TGF-beta-induced EMT. Further analysis of cofilin1 and beta-actin revealed an increase in their mRNA stability in response to TGF-beta, contributing to the observed increase in mRNA and protein expression. These results are the first demonstration of post-transcriptional regulation of cytoskeletal remodelling and a key role for cofilin1 during TGF-beta-induced EMT.
Journal of Proteome Research 02/2009; 8(1):35-47. · 5.11 Impact Factor
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Venkateshwar G. Keshamouni,
Pratik Jagtap,
George Michailidis,
John R. Strahler,
Rork Kuick,
Ajaya Kumar Reka,
Panagiotis Papoulias,
Rashmi Krishnapuram,
Anjaiah Srirangam,
Theodore J. Standiford, Philip C. Andrews,
Gilbert S. Omenn
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ABSTRACT: To gain insights into how TGF-β regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, including newly associated proteins calpain, fascin and macrophage-migration inhibitory factor (MIF). Comparing protein and mRNA expression overtime showed that all the 14 up-regulated proteins involved in the actin-cytoskeleton remodeling were accompanied by increases in corresponding mRNA expression. Interestingly, siRNA mediated knockdown of cofilin1 potentiated TGF-β-induced EMT. Further analysis of cofilin1 and β-actin revealed an increase in their mRNA stability in response to TGF-β, contributing to the observed increase in mRNA and protein expression. These results are the first demonstration of post-transcriptional regulation of cytoskeletal remodelling and a key role for cofilin1 during TGF-β-induced EMT.Keywords: iTRAQ; quatitative proteomics; TGF-β; lung cancer; epithelial-mesenchymal transition; actin-cytoskeleton remodeling; beta-actin; cofilin1; calpain; ezrin; moesin
01/2009;
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ABSTRACT: Current mass spectrometers provide a number of alternative methodologies for producing tandem mass spectra specifically for phosphopeptide analysis. In particular, generation of MS(3) spectra in a data-dependent manner upon detection of the neutral loss of a phosphoric acid in MS(2) spectra is a popular technique for circumventing the problem of poor phosphopeptide backbone fragmentation. The newer Multistage Activation method provides another option. Both these strategies require additional cycle time on the instrument and therefore reduce the number of spectra that can be measured in the same amount of time. Additional informatics is often required to make most efficient use of the additional information provided by these spectra as well. This work presents a comparison of several commonly used mass spectrometry methods for the study of phosphopeptide-enriched samples: an MS(2)-only method, a Multistage Activation method, and an MS(2)/MS(3) data-dependent neutral loss method. Several strategies for dealing effectively with the resulting MS(3) data in the latter approach are also presented and compared. The overall goal is to infer whether any one methodology performs significantly better than another for identifying phosphopeptides. On data presented here, the Multistage Activation methodology is demonstrated to perform optimally and does not result in significant loss of unique peptide identifications.
Journal of Proteome Research 01/2009; 8(2):887-99. · 5.11 Impact Factor
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ABSTRACT: Current mass spectrometers provide a number of alternative methodologies for producing tandem mass spectra specifically for phosphopeptide analysis. In particular, generation of MS3 spectra in a data-dependent manner upon detection of the neutral loss of a phosphoric acid in MS2 spectra is a popular technique for circumventing the problem of poor phosphopeptide backbone fragmentation. The newer Multistage Activation method provides another option. Both these strategies require additional cycle time on the instrument and therefore reduce the number of spectra that can be measured in the same amount of time. Additional informatics is often required to make most efficient use of the additional information provided by these spectra as well. This work presents a comparison of several commonly used mass spectrometry methods for the study of phosphopeptide-enriched samples: an MS2-only method, a Multistage Activation method, and an MS2/MS3 data-dependent neutral loss method. Several strategies for dealing effectively with the resulting MS3 data in the latter approach are also presented and compared. The overall goal is to infer whether any one methodology performs significantly better than another for identifying phosphopeptides. On data presented here, the Multistage Activation methodology is demonstrated to perform optimally and does not result in significant loss of unique peptide identifications.Keywords: Protein phosphorylation; mass spectrometry; MS3; Multistage Activation; phosphoproteomics; bioinformatics; peptide identification; database search
12/2008;
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Newaj M Abdullah,
Maureen Kachman,
Angela Walker,
Angela E Hawley,
Shriley K Wrobleski,
Daniel D Myers,
John R Strahler, Philip C Andrews,
Goerge C Michailidis,
Eduardo Ramacciotti,
Peter K Henke,
Thomas W Wakefield
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ABSTRACT: Microparticles are small membrane vesicles released from activated cells and are associated with thrombosis and inflammation. Microparticle contain a unique subset of surface protein derived form the parent cell and may be responsible for their diverse biological functions. To identify these proteins, juvenile baboons (Papio anubis, n = 4) underwent iliac vein thrombosis with 6-hour balloon occlusion. Plasma samples were taken at baselines and at 2 days postthrombosis for microparticle analysis. Microparticles were extracted from platelet-poor plasma, digest separately with trypsin and tagged using isobaric tagging for relative and absolute quantitation reagents. The digests were subjected to 2-dimensional liquid chromatographic separation followed by matrix-assisted laser desorption/ionization tandem mass spectrometry. Peak lists were generated and searched against all primate sequences. For protein identity, a minimum of 2 peptides at 95% confidence interval was required. Later, isobaric tagging for relative and absolute quantitation ratios were generated comparing relative protein level of day 2 to baseline. The proteomic analysis was performed twice for each blood sample, totaling 8 experiments. Proteins were considered elevated of depressed if the isobaris tagging for relative and absolute quantitation ratio deviated by 20% changes from normal and a P value less than .05. Significantly, 7 proteins were differentially expressed on day 2 compared to baseline, and appeared in at least 3 animals and regulated in at least 4 experiment. Among these 7 proteins, upregulated proteins include various forms of fibrinogen and alpha-1-antichymotrypsin and downregulated proteins include immunoglobulins. These proteins influence thrombosis and inflammation through hemostatic plug formation (fibrinogen), inhibiting neutrophil adhesion (alpha-1-antichymoptrypsin), and immunoregulation (immunoglobulins). Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis.
Clinical and Applied Thrombosis/Hemostasis 12/2008; 15(2):201-8. · 1.33 Impact Factor
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ABSTRACT: The zymogen granule is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and is a classic model for studying secretory granule function. Our long term goal is to develop a comprehensive architectural model for zymogen granule membrane (ZGM) proteins that would direct new hypotheses for subsequent functional studies. Our initial proteomics analysis focused on identification of proteins from purified ZGM (Chen, X., Walker, A. K., Strahler, J. R., Simon, E. S., Tomanicek-Volk, S. L., Nelson, B. B., Hurley, M. C., Ernst, S. A., Williams, J. A., and Andrews, P. C. (2006) Organellar proteomics: analysis of pancreatic zymogen granule membranes. Mol. Cell. Proteomics 5, 306-312). In the current study, a new global topology analysis of ZGM proteins is described that applies isotope enrichment methods to a protease protection protocol. Our results showed that tryptic peptides of ZGM proteins were separated into two distinct clusters according to their isobaric tag for relative and absolute quantification (iTRAQ) ratios for proteinase K-treated versus control zymogen granules. The low iTRAQ ratio cluster included cytoplasm-orientated membrane and membrane-associated proteins including myosin V, vesicle-associated membrane proteins, syntaxins, and all the Rab proteins. The second cluster having unchanged ratios included predominantly luminal proteins. Because quantification is at the peptide level, this technique is also capable of mapping both cytoplasm- and lumen-orientated domains from the same transmembrane protein. To more accurately assign the topology, we developed a statistical mixture model to provide probabilities for identified peptides to be cytoplasmic or luminal based on their iTRAQ ratios. By implementing this approach to global topology analysis of ZGM proteins, we report here an experimentally constrained, comprehensive topology model of identified zymogen granule membrane proteins. This model contributes to a firm foundation for developing a higher order architecture model of the ZGM and for future functional studies of individual ZGM proteins.
Molecular & Cellular Proteomics 09/2008; 7(12):2323-36. · 7.40 Impact Factor
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ABSTRACT: Assembly of adenovirus particles is thought to be similar to that of bacteriophages, in which the double-stranded DNA genome is inserted into a preformed empty capsid. Previous studies from our and other laboratories have implicated the viral IVa2 protein as a key component of the encapsidation process. IVa2 binds to the packaging sequence on the viral chromosome in a sequence-specific manner, alone and in conjunction with the viral L4 22K protein. In addition, it interacts with the viral L1 52/55-kDa protein, which is required for DNA packaging. Finally, a mutant virus that does not produce IVa2 is unable to produce any capsids. Therefore, it has been proposed that IVa2 nucleates capsid assembly. A prediction of such a model is that the IVa2 protein would be found at a unique vertex of the mature virion. In this study, the location of IVa2 in the virion has been analyzed using immunogold staining and electron microscopy, and the copy number of IVa2 in virions was determined using three independent methods, quantitative mass spectrometry, metabolic labeling, and Western blotting. The results indicate that it resides at a unique vertex and that there are approximately six to eight IVa2 molecules in each particle. These findings support the hypothesis that the IVa2 protein plays multiple roles in the viral assembly process.
Journal of Virology 08/2008; 82(18):9086-93. · 5.40 Impact Factor
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ABSTRACT: Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO2) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well. These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and also increase sample complexity. We show here that the conversion of peptide carboxylates to their corresponding methyl esters sharply reduces nonspecific binding, improving the selectivity for phosphopeptides, just as has been reported for immobilized metal affinity chromatography (IMAC) columns. We also present evidence that monophosphorylated peptides can be effectively fractionated from multiply phosphorylated peptides, as well as acidic peptides, via stepwise elution from TiO2 using pH step gradients from pH 8.5 to pH 11.5. These approaches were applied to human CDK2 phosphorylated in vitro by yeast CAK1p in the absence of cyclin. We confirmed phosphorylation at T160, a site previously documented and shown to be necessary for CDK2 activity. However, we also discovered several novel sites of partial phosphorylation at S46, T47, T165, and Y168 when ion-suppressing nonphosphorylated peptides were eliminated using the new protocols.
Analytical Biochemistry 07/2008; 377(2):234-42. · 3.00 Impact Factor
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ABSTRACT: Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H(2)O(2)) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30-90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H(2)O(2) or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.
Proceedings of the National Academy of Sciences 07/2008; 105(24):8197-202. · 9.68 Impact Factor
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ABSTRACT: A FASTA file archive and reference resource has been added to ProteomeCommons.org. Motivation for this new functionality derives from two primary sources. The first is the recent FASTA standardization work done by the Human Proteome Organization's Proteomics Standards Initiative (HUPO-PSI). Second is the general lack of a uniform mechanism to properly cite FASTA files used in a study, and to publicly access such FASTA files post-publication. An extension to the Tranche data sharing network has been developed that includes web-pages, documentation, and tools for facilitating the use of FASTA files. These include conversion to the new HUPO-PSI format, and provisions for both citing and publicly archiving FASTA files. This new resource is available immediately, free of charge, and can be accessed at http://www.proteomecommons.org/data/fasta/. Source-code for related tools is also freely available under the BSD license.
Proteomics 06/2008; 8(9):1756-7. · 4.43 Impact Factor
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ABSTRACT: Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on non-redundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation.
Molecular & Cellular Proteomics 05/2008; 7(4):750-67. · 7.40 Impact Factor
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Suresh Mathivanan,
Mukhtar Ahmed,
Natalie G Ahn,
Hainard Alexandre,
Ramars Amanchy, Philip C Andrews,
Joel S Bader,
Brian M Balgley,
Marcus Bantscheff,
Keiryn L Bennett, [......],
Xiaoyue Wang,
Stefan Wiemann,
Billy Wu,
Tao Xu,
John R Yates,
Jun Zhong,
Ming Zhou,
Yunping Zhu,
Petra Zurbig,
Akhilesh Pandey
Nature Biotechnology 03/2008; 26(2):164-7. · 29.50 Impact Factor
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ABSTRACT: Methods combining the high throughput and sensitivity of matrix-assisted laser desorption ionization mass spectometry (MALDI-MS)
with polyacrylamide gel electrophoresis (PAGE) are rapidly gaining attention, but almost exclusively for applications eluting
proteolytic digest products from gels and membranes (1–9). These methods fulfill a considerable need for identifying unknown proteins isolated in polyacrylamide gels, but they do
not provide masses for intact proteins, a particular concern should the goal be the understanding of anomalous electrophoretic
migrations, characterizing the complete ensemble of posttranslational modifications displayed by proteins, or understanding
why multiple protein spots on a two-dimensional (2-D) gel are all identified as the same protein. These issues are particularly
relevant for low-abundance proteins, which may be identified by matching the masses of only a few tryptic peptides to those
predicted from cleavage of an unmodified protein, or by matching small stretches of sequence information (9,10). In some cases, those matches may correspond to <10% of the total protein sequence (9). Analysis of intact proteins has been performed following electroblotting to membranes (11–19), but we will describe a method to link gel electrophoresis directly to MALDI-MS, yielding masses of both intact proteins
and cleavage products without electroelution or electroblotting (20–22). Key to the success of this approach are ultrathin polyacrylamide gels, which dry to thicknesses of 10 μm or less and which
have the additional advantages of rapid preparation and electrophoresis run times.
02/2008: pages 473-485;
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ABSTRACT: Pancreatic zymogen granules (ZGs) are specialized for digestive enzyme storage and regulated secretion in exocrine pancreas and are a classical model for studying secretory granule function. To understand the function of this organelle, we have conducted a proteomic study to identify the ZG membrane (ZGM) proteins from ZGs purified by Percoll gradient centrifugation. By combining multiple separation strategies including two-dimensional gel electrophoresis and two-dimensional HPLC with tandem mass spectrometry, we identified 101 proteins from purified ZGMs including a large number of proteins previously unknown on ZGMs. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomics strategy was used to measure the enrichment of intrinsic membrane proteins through the purification steps by labeling crude, KBr-, and Na(2)CO(3)-washed ZGMs with multiplexed isobaric tags (iTRAQtrade mark), 114, 116, and 117, respectively. The proteins with 117:114 ratios greater than one correlated well with intrinsic membrane proteins that contain either known or predicted transmembrane domains.
Methods in molecular biology (Clifton, N.J.) 02/2008; 432:275-87.
