Proteomics

Protein interaction networks in medicine and disease.

Publication Date: 2012 May 16 PMID: 22593007
Authors: Taylor, I. W. - Wrana, J. L.
Journal: Proteomics

The physical interaction of proteins is subject to intense investigation that has revealed that proteins are assembled into large densely connected networks. In this review, we will examine how signaling pathways can be combined to form higher order protein interaction networks. By using network graph theory, these interaction networks can be further analyzed for global organization, which has revealed unique aspects of the relationships between protein networks and complex biological phenotypes. Moreover, several studies have shown that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer progression. These relationships suggest a novel paradigm for treatment of complex multigenic disease where the protein interaction network is the target of therapy more so than individual molecules within the network.

post to: CiteULike

Computational structural analysis of protein interactions and networks.

Publication Date: 2012 May 16 PMID: 22593000
Authors: Hooda, Y. - Kim, P. M.
Journal: Proteomics

Protein interactions have been at the focus of computational biology in recent years. In particular, interest has come from two different communities-structural and systems biology. Here, we will discuss key systems and structural biology methods that have been used for analysis and prediction of protein-protein interactions and the insight these approaches have provided on the nature and organization of protein-protein interactions inside cells.

post to: CiteULike

Two steps forward-one step back: Advances in affinity purification mass spectrometry of macromolecular complexes.

Publication Date: 2012 May 16 PMID: 22592981
Authors: Oeffinger, M.
Journal: Proteomics

Cellular functions are defined by the dynamic interactions of proteins within macromolecular networks. Deciphering these complex interplays is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems, from a simple bacterial cell to highly regulated neuronal cells or cancerous tissue. In the last decade, affinity purification (AP) coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their macromolecular assemblies. This review discusses recent advances in AP approaches, from cell lysis to the importance of sample preparation and the choice of AP matrix as well as the development of different epitope tags and strategies to study dynamic interactions, with an emphasis on RNA-protein interaction networks.

post to: CiteULike

Interactome mapping for analysis of complex phenotypes: Insights from benchmarking binary interaction assays.

Publication Date: 2012 May 16 PMID: 22589225
Authors: Braun, P.
Journal: Proteomics

Protein interactions mediate essentially all biological processes and analysis of protein-protein interactions using both large-scale and small-scale approaches has contributed fundamental insights to the understanding of biological systems. In recent years, interactome network maps have emerged as an important tool for analyzing and interpreting genetic data of complex phenotypes. Complementary experimental approaches to test for binary, direct interactions, and for membership in protein complexes are used to explore the interactome. The two approaches are not redundant but yield orthogonal perspectives onto the complex network of physical interactions by which proteins mediate biological processes. In recent years, several publications have demonstrated that interactions from high-throughput experiments can be equally reliable as the high quality subset of interactions identified in small-scale studies. Critical for this insight was the introduction of standardized experimental benchmarking of interaction and validation assays using reference sets. The data obtained in these benchmarking experiments have resulted in greater appreciation of the limitations and the complementary strengths of different assays. Moreover, benchmarking is a central element of a conceptual framework to estimate interactome sizes and thereby measure progress toward near complete network maps. These estimates have revealed that current large-scale data sets, although often of high quality, cover only a small fraction of a given interactome. Here, I review the findings of assay benchmarking and discuss implications for quality control, and for strategies toward obtaining a near-complete map of the interactome of an organism.

post to: CiteULike

Combining many interaction networks to predict gene function and analyze gene lists.

Publication Date: 2012 May 16 PMID: 22589215
Authors: Mostafavi, S. - Morris, Q.
Journal: Proteomics

post to: CiteULike

Cover picture: proteomics 9'12.

Publication Date: 2012 May PMID: 22589196
Authors:
Journal: Proteomics

post to: CiteULike

Editorial board: proteomics.

Publication Date: 2012 May PMID: 22589195
Authors:
Journal: Proteomics

post to: CiteULike

Contents - proteomics 9'12.

Publication Date: 2012 May PMID: 22589194
Authors:
Journal: Proteomics

post to: CiteULike

Proteomic analysis of the microenvironment of developing oocytes.

Publication Date: 2012 May PMID: 22589193
Authors: Twigt, J. - Steegers-Theunissen, R. P. - Bezstarosti, K. - Demmers, J. A.
Journal: Proteomics

We utilized a setup based on extensive pre-fractionation of proteolytic peptides and nanoflow reversed-phase LC-MS/MS to identify the (sub)proteome of human follicular fluid (FF). In this in-depth screen, 246 specific proteins were identified, the majority of which are involved in coagulation- and immune-response pathways. Our aim is to define a set of FF protein markers, which could predict oocyte quality.

post to: CiteULike

Comprehensive identification of novel post-translational modifications in cellular peroxiredoxin 6.

Publication Date: 2012 May PMID: 22589192
Authors: Jeong, J. - Kim, Y. - Kyung Seong, J. - Lee, K. J.
Journal: Proteomics

Peroxiredoxin 6 (PRDX6), a 1-Cys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post-translational modifications (PTMs) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTMs in PRDX6 utilizing proteomic tools including nanoUPLC-ESI-q-TOF MS/MS employing selectively excluded mass screening analysis (SEMSA) in conjunction with MOD(i) and MODmap algorithm. We chose PRDX6 obtained from liver tissues from two inbred mouse strains, C57BL/6J and C3H/HeJ, which vary in their susceptibility to high-fat diet-induced obesity and atherosclerosis, and a B16F10 melanoma cell line for this study. When PRDX6 protein samples were separated on 2D-PAGE based on pI, several PRDX6 spots appeared. They were purified and the low abundant PTMs in each PRDX6 spot were analyzed. Unexpected mass shifts (Deltam = -34, +25, +64, +87, +103, +134, +150, +284 Da) observed at active site cysteine residue (Cys47) were quantified using precursor ion intensities. Mass differences of -34, +25, and +64 Da are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and Cys-SO(2) -SH, respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da) as well as unknown modifications (+134, +150, +284 Da). Comprehensive analysis of these PTMs revealed that the PRDX6 exists as a heterogeneous mixture of molecules containing a multitude of PTMs. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX6 from mouse liver tissues included, among others, mono- and dioxidation at Trp and Met, acetylation at Lys, and deamidation at Asn and Gln. Comprehensive identification of the diverse PTMs occurring in this bifunctional PRDX6 enzyme should help understand how PRDX6 plays key roles in oxidative stresses.

post to: CiteULike

Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet.

Publication Date: 2012 May PMID: 22589191
Authors: Thomas, A. - Stevens, A. P. - Klein, M. S. - Hellerbrand, C. - Dettmer, K. - Gronwald, W. - Oefner, P. J. - Reinders, J.
Journal: Proteomics

Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed. Hepatic steatosis manifested itself after 2 days of feeding, albeit significant changes in the liver-soluble proteome were not evident before day 10 in the absence of inflammatory or fibrotic signs. Proteomic alterations affected mainly energy and amino acid metabolism, detoxification processes, urea cycle, and the one-carbon/S-adenosylmethionine pathways. Additionally, intermediates of relevant affected pathways were quantified from liver tissue, confirming the findings from the proteomic analysis.

post to: CiteULike

Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilus HB8.

Publication Date: 2012 May PMID: 22589190
Authors: Takahata, Y. - Inoue, M. - Kim, K. - Iio, Y. - Miyamoto, M. - Masui, R. - Ishihama, Y. - Kuramitsu, S.
Journal: Proteomics

We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram-negative eubacterium Thermus thermophilus HB8 using gel-free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 A of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilus HB8.

post to: CiteULike

Proteogenomic evidence for beta-oxidation of plant-derived 3-phenylpropanoids in "Aromatoleum aromaticum" EbN1.

Publication Date: 2012 May PMID: 22589189
Authors: Trautwein, K. - Wilkes, H. - Rabus, R.
Journal: Proteomics

The betaproteobacterium "Aromatoleum aromaticum" EbN1 utilizes eight different plant-derived nonhydroxylated (e.g. cinnamate) and hydroxylated (e.g. p-coumarate) 3-phenylpropanoids with nitrate as electron acceptor. Differential protein profiling (2D-DIGE) revealed abundance increases of five proteins (EbA5316 to EbA5320) during anaerobic growth with cinnamate, hydrocinnamate, p-coumarate, and 3-(4-hydroxyphenyl)propanoate, compared to anaerobic benzoate-adapted cells serving as reference state. The predicted functions of four of these proteins (EbA5317, fatty acid-coenzyme A (CoA) ligase; EbA5318, enoyl-CoA hydratase/isomerase; EbA5319, beta-ketothiolase; and EbA5320, 3-hydroxyacyl-CoA dehydrogenase) suggest beta-oxidation of the above 3-phenylpropanoids to benzoyl-CoA and p-hydroxybenzoyl-CoA, respectively. The fifth protein (EbA5316, ABC-type periplasmic solute-binding protein) could be involved in 3-phenylpropanoid uptake. The detection of 3-hydroxy-3-phenylpropanoate during anaerobic growth with cinnamate and hydrocinnamate or 3-hydroxy-3-(4-hydroxyphenyl)propanoate during anaerobic growth with p-coumarate and 3-(4-hydroxyphenyl)propanoate supports the proteome-predicted beta-oxidation pathway. Based on the specific formation of EbA5316-20 also during anaerobic growth with further 3-phenylpropanoid growth substrates including cinnamyl alcohol, m-coumarate, 3-(3,4-dihydroxyphenyl)propanoate and 3,4-dihydroxycinnamate (caffeate), a common beta-oxidation route is proposed for 3-phenylpropanoid degradation in strain EbN1. The low amount of metabolites attributable to cometabolic transformation of nongrowth supporting 3-phenylpropanoids (e.g. o-coumarate, ferulate) may be indicative for a high substrate specificity of the involved enzymes.

post to: CiteULike

Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics.

Publication Date: 2012 May PMID: 22589188
Authors: Denoncin, K. - Schwalm, J. - Vertommen, D. - Silhavy, T. J. - Collet, J. F.
Journal: Proteomics

beta-Barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, beta-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all beta-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the nonviable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli.

post to: CiteULike

Protein alteration of HepG2.2.15 cells induced by iron overload.

Publication Date: 2012 May PMID: 22589187
Authors: Fang, C. - Zhao, C. - Liu, X. - Yang, P. - Lu, H.
Journal: Proteomics

Hepatitis B can progress into hepatocellular carcinoma. Body irons may interfere with the clearance of hepatitis B virus (HBV) and contribute to genesis of tumor. To investigate the role of iron played in HBV-related pathogenesis, here we studied the effect of iron with different concentrations and valence states on growth of HepG2.2.15 cells and secretion of virus proteins. A strong tolerance of HepG2.2.15 cells to iron challenge was found. The concentration of hepatitis B surface antigen in cell culture medium was decreased after iron stimulation. Lower concentrations of iron facilitated hepatitis B e-antigen (HBeAg) secretion. Fe(2+) appeared more effective on HBeAg secretion than Fe(3+) did. In parallel, the differential protein profiles in HepG2.2.15 cells were studied by iTRAQ and LC-MS/MS. The differentially expressed proteins were mainly involved in stress response, signal transduction, apoptosis, etc. Four proteins (14-3-3 beta/alpha, VCP, migration inhibitory factor, and Nup153) were verified by Western-blotting and found to be consistent with the iTRAQ data. Interestingly, nuclear import of Nuclear factor kappa B (NFkappaB) and its activity were found to be affected by the decreased Nup153 in iron stimulated HepG2.2.15 cells. The results may indicate possible molecular mechanism how the synergism of HBV and iron stimulation damages host liver cells.

post to: CiteULike

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells.

Publication Date: 2012 May PMID: 22589186
Authors: Lochab, S. - Pal, P. - Kanaujiya, J. K. - Tripathi, S. B. - Kapoor, I. - Bhatt, M. L. - Sanyal, S. - Behre, G. - Trivedi, A. K.
Journal: Proteomics

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.

post to: CiteULike

Impaired ubiquitin-proteasome-mediated PGC-1alpha protein turnover and induced mitochondrial biogenesis secondary to complex-I deficiency.

Publication Date: 2012 May PMID: 22589185
Authors: Farhoud, M. H. - Nijtmans, L. G. - Wanders, R. J. - Wessels, H. J. - Lasonder, E. - Janssen, A. J. - Rodenburg, R. R. - van den Heuvel, L. P. - Smeitink, J. A.
Journal: Proteomics

Most eukaryotic cells depend on mitochondrial OXidative PHOSphorylation (OXPHOS) in their ATP supply. The cellular consequences of OXPHOS defects and the pathophysiological mechanisms in related disorders are incompletely understood. Using a quantitative proteomics approach we provide evidence that a genetic defect of complex-I of the OXPHOS system may associate with transcriptional derangements of mitochondrial biogenesis through stabilization of the master transcriptional regulator PPARgamma co-activator 1alpha (PGC-1alpha) protein. Chronic oxidative stress suppresses the gene expression of PGC-1alpha but concomitant inhibition of the ubiquitin-proteasome system (UPS) can stabilize this co-activator protein, thereby inducing its downstream metabolic gene expression programs. Thus, mitochondrial biogenesis, which lays at the heart of the homeostatic control of energy metabolism, can be deregulated by secondary impairments of the protein turnover machinery.

post to: CiteULike

MALDI-TOF-MS analysis of sialylated glycans and glycopeptides using 4-chloro-alpha-cyanocinnamic acid matrix.

Publication Date: 2012 May PMID: 22589184
Authors: Selman, M. H. - Hoffmann, M. - Zauner, G. - McDonnell, L. A. - Balog, C. I. - Rapp, E. - Deelder, A. M. - Wuhrer, M.
Journal: Proteomics

For MALDI analysis of glycans and glycopeptides, the choice of matrix is crucial in minimizing desialylation by mass spectrometric in-source and metastable decay. Here, we evaluated the potential of 4-chloro-alpha-cyanocinnamic acid (Cl-CCA) for MALDI-TOF-MS analysis of labile sialylated tryptic N-glycopeptides and released N- and O-glycans. Similar to DHB, but in contrast to CHCA, the Cl-CCA matrix allowed the analysis of sialylated N-glycans and glycopeptides in negative ion mode MALDI-TOF-MS. Dried droplet preparations of Cl-CCA provided microcrystals with a homogeneous spatial distribution and high shot-to-shot repeatability similar to CHCA, which simplified the automatic measurement and improved the resolution and mass accuracy. Interestingly, reflectron-positive ion mode analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled O-glycans with Cl-CCA revealed more complete profiles than with DHB and CHCA. In conclusion, we clearly demonstrate the high potential of this rationally designed matrix for glycomics and glycoproteomics.

post to: CiteULike

PRDB: Protein Repeat DataBase.

Publication Date: 2012 May PMID: 22589183
Authors: Jorda, J. - Baudrand, T. - Kajava, A. V.
Journal: Proteomics

Rapidly increasing genomic data present new challenges for scientists: making sense of millions of amino acid sequences requires a systematic approach and information about their 3D structure, function, and evolution. Over the last decade, numerous studies demonstrated the fundamental importance of protein tandem repeats and their involvement in human diseases. Bioinformatics analysis of these regions requires special computer programs and databases, since the conventional approaches predominantly developed for globular domains have limited success. To perform a global comparative analysis of protein tandem repeats, we developed the Protein Tandem Repeat DataBase (PRDB). PRDB is a curated database that includes the protein tandem repeats found in sequence databanks by the T-REKS program. The database is available at http://bioinfo.montp.cnrs.fr/?r=repeatDB.

post to: CiteULike

Ultra-high intra-spectrum mass accuracy enables unambiguous identification of fragment reporter ions in isobaric multiplexed quantitative proteomics.

Publication Date: 2012 May PMID: 22589182
Authors: Pachl, F. - Fellenberg, K. - Wagner, C. - Kuster, B.
Journal: Proteomics

Isobaric tagging using reagents such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ) have become popular tools for mass spectrometry based quantitative proteomics. Because the peptide quantification information is collected in tandem mass spectra, the accuracy and precision of this method largely depend on the resolution with which precursor ions can be selected for the fragmentation and the specificity of the generated reporter ion. The latter can constitute an issue if near isobaric ion signals are present in such spectra because they may distort quantification results. We propose a simple remedy for this problem by identifying reporter ions via the accurate mass differences within a single tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra. Our results show that this leads to unambiguous reporter ion identification and complete removal of interfering signals. This mode of data processing is easily implemented in software and offers advantages for protein quantification based on few peptides.

post to: CiteULike

An improved quantitative mass spectrometry analysis of tumor specific mutant proteins at high sensitivity.

Publication Date: 2012 May PMID: 22589181
Authors: Ruppen-Canas, I. - Lopez-Casas, P. P. - Garcia, F. - Ximenez-Embun, P. - Munoz, M. - Morelli, M. P. - Real, F. X. - Serna, A. - Hidalgo, M. - Ashman, K.
Journal: Proteomics

New disease specific biomarkers, especially for cancer, are urgently needed to improve individual diagnosis, prognosis, and treatment selection, that is, for personalized medicine. Genetic mutations that affect protein function drive cancer. Therefore, the detection of such mutations represents a source of cancer specific biomarkers. Here we confirm the implementation of the mutant protein specific immuno-SRM (where SRM is selective reaction monitoring) mass spectrometry method of RAS proteins reported by Wang et al. [Proc. Natl. Acad. Sci. USA 2011, 108, 2444-2449], which exploits an antibody to simultaneously capture the different forms of the target protein and the resolving power and sensitivity of LC-MS/MS and improve the technique by using a more sensitive mass spectrometer. The mutant form G12D was quantified by SRM on a QTRAP 5500 mass spectrometer and the MIDAS workflow was used to confirm the sequence of the targeted peptides. This assay has been applied to quantify wild type and mutant RAS proteins in patient tumors, xenografted human tissue, and benign human epidermal tumors at high sensitivity. The limit of detection for the target proteins was as low as 12 amol (0.25 pg). It requires low starting amounts of tissue (ca.15 mg) that could be obtained from a needle aspiration biopsy. The described strategy could find application in the clinical arena and be applied to the study of expression of protein variants in disease.

post to: CiteULike

Digital microfluidic hydrogel microreactors for proteomics.

Publication Date: 2012 May PMID: 22589180
Authors: Luk, V. N. - Fiddes, L. K. - Luk, V. M. - Kumacheva, E. - Wheeler, A. R.
Journal: Proteomics

Proteolytic digestion is an essential step in proteomic sample processing. While this step has traditionally been implemented in homogeneous (solution) format, there is a growing trend to use heterogeneous systems in which the enzyme is immobilized on hydrogels or other solid supports. Here, we introduce the use of immobilized enzymes in hydrogels for proteomic sample processing in digital microfluidic (DMF) systems. In this technique, preformed cylindrical agarose discs bearing immobilized trypsin or pepsin were integrated into DMF devices. A fluorogenic assay was used to optimize the covalent modification procedure for enzymatic digestion efficiency, with maximum efficiency observed at 31 mug trypsin in 2-mm diameter agarose gel discs. Gel discs prepared in this manner were used in an integrated method in which proteomic samples were sequentially reduced, alkylated, and digested, with all sample and reagent handling controlled by DMF droplet operation. Mass spectrometry analysis of the products revealed that digestion using the trypsin gel discs resulted in higher sequence coverage in model analytes relative to conventional homogenous processing. Proof-of-principle was demonstrated for a parallel digestion system in which a single sample was simultaneously digested on multiple gel discs bearing different enzymes. We propose that these methods represent a useful new tool for the growing trend toward miniaturization and automation in proteomic sample processing.

post to: CiteULike

Enhanced identification of peptides lacking basic residues by LC-ESI-MS/MS analysis of singly charged peptides.

Publication Date: 2012 May PMID: 22589179
Authors: Biniossek, M. L. - Schilling, O.
Journal: Proteomics

Peptide sequences lacking basic residues (arginine, lysine, or histidine, referred to as "base-less") are of particular importance in proteomic experiments targeting protein C-termini or employing nontryptic proteases such as GluC or chymotrypsin. We demonstrate enhanced identification of base-less peptides by focused analysis of singly charged precursors in liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Singly charged precursors are often excluded from fragmentation and sequence analysis in LC-MS/MS. We generated different pools of base-less and base-containing peptides by tryptic and nontryptic digestion of bacterial proteomes. Focused LC-MS/MS analysis of singly charged precursor ions yielded predominantly base-less peptide identifications. Similar numbers of base-less peptides were identified by LC-MS/MSanalysis targeting multiply charged precursors. There was little redundancy between the base-less sequences derived by both MS/MSschemes. In the present experimental outcome, additional LC-MS/MSanalysis of singly charged precursors substantially increased the identification rate of base-less sequences derived from multiply charged precursors. In conclusion, LC-MS/MSbased identification of base-less peptides is substantially enhanced by additional focused analysis of singly charged precursors.

post to: CiteULike

PROTEOMICS - Clinical Applications Table of Contents: Volume 6, Issue 1-2 Special Issue: Reviews 2012 Editor: Michael J. Dunn.

Publication Date: 2012 May PMID: 22589178
Authors:
Journal: Proteomics

post to: CiteULike

In this issue.

Publication Date: 2012 May PMID: 22589177
Authors:
Journal: Proteomics

post to: CiteULike

A quantitative proteomic analysis of lung epithelial (A549) cells infected with 2009 pandemic influenza A virus using stable isotope labelling with amino acids in cell culture.

Publication Date: 2012 May PMID: 22585751
Authors: Dove, B. K. - Surtees, R. - Bean, T. J. - Munday, D. - Wise, H. M. - Digard, P. - Carroll, M. W. - Ajuh, P. - Barr, J. N. - Hiscox, J. A.
Journal: Proteomics

Influenza A virus is one of the world's major uncontrolled pathogens, causing seasonal epidemics as well as global pandemics. This was evidenced by the recent emergence and now prevalence of the 2009 swine origin pandemic H1N1 influenza A virus. In this study, quantitative proteomics using stable isotope labelling with amino acids in cell culture was used to investigate the changes in the host cell proteome in cells infected with pandemic H1N1 influenza A virus. The study was conducted in A549 cells that retain properties similar to alveolar cells. Several global pathways were affected, including cell cycle regulation and lipid metabolism, and these could be correlated with recent microarray analyses of cells infected with influenza A virus. Taken together, both quantitative proteomics and transcriptomic approaches can be used to identify potential cellular proteins whose functions in the virus life cycle could be targeted for chemotherapeutic intervention.

post to: CiteULike