http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2013 , Hindawi Publishing Corporation . All rights reserved. Mon, 29 Apr 2013 11:14:52 +0000 http://www.hindawi.com/journals/ijpro/2013/621029/ Mu Wang, Bomie Han, and Valerie Wasinger Copyright © 2013 Mu Wang et al. All rights reserved. Tue, 23 Apr 2013 08:36:22 +0000 http://www.hindawi.com/journals/ijpro/2013/674282/ Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference. Richard E. Higgs, Jon P. Butler, Bomie Han, and Michael D. Knierman Copyright © 2013 Richard E. Higgs et al. All rights reserved. Mon, 22 Apr 2013 12:50:22 +0000 http://www.hindawi.com/journals/ijpro/2013/135709/ The ontogenetic variability in venom composition of some snake genera, including Bothrops, as well as the biological implications of such variability and the search of new molecules that can neutralize the toxic components of these venoms have been the subject of many studies. Thus, considering the resistance of Bothrops jararaca to the toxic action of its own venom and the ontogenetic variability in venom composition described in this species, a comparative study of the plasma composition of juvenile and adult B. jararaca snakes was performed through a proteomic approach based on 2D electrophoresis and mass spectrometry, which allowed the identification of proteins that might be present at different levels during ontogenetic development. Among the proteins identified by mass spectrometry, antihemorrhagic factor Bj46a was found only in adult plasma. Moreover, two spots identified as phospholipase A2 inhibitors were significantly increased in juvenile plasma, which can be related to the higher catalytic PLA2 activity shown by juvenile venom in comparison to that of adult snakes. This work shows the ontogenetic variability of B. jararaca plasma, and that these changes can be related to the ontogenetic variability described in its venom. Karen de Morais-Zani, Kathleen Fernandes Grego, Aparecida Sadae Tanaka, and Anita Mitico Tanaka-Azevedo Copyright © 2013 Karen de Morais-Zani et al. All rights reserved. Thu, 04 Apr 2013 09:45:29 +0000 http://www.hindawi.com/journals/ijpro/2013/791985/ The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs) are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM) mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH) was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation. Jason M. Held, Birgit Schilling, Alexandria K. D'Souza, Tara Srinivasan, Jessica B. Behring, Dylan J. Sorensen, Christopher C. Benz, and Bradford W. Gibson Copyright © 2013 Jason M. Held et al. All rights reserved. Mon, 11 Mar 2013 15:30:40 +0000 http://www.hindawi.com/journals/ijpro/2013/581862/ Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or label-free methods were used, and the resulting samples were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). To reduce a possible bias from quantitation software, we applied several software packages for each procedure. ProteinPilot and Scaffold Q+ software were used for iTRAQ-labeled samples, while Progenesis LC-MS and ProgenesisF-T2PQ/T3PQ were employed for label-free analyses. R2 correlation coefficients correlated well between two software packages applied to the same datasets with values between 0.48 and 0.78 for iTRAQ-label-based quantitations and 0.5 and 0.86 for label-free quantitations. Analyses of label-free samples showed higher levels of protein up- or downregulation in comparison to iTRAQ-labeled samples. The concentration differences were further evaluated by Western blotting for four downregulated proteins. These data suggested that the label-free method was more accurate than the iTRAQ method. Hung V. Trinh, Jonas Grossmann, Peter Gehrig, Bernd Roschitzki, Ralph Schlapbach, Urs F. Greber, and Silvio Hemmi Copyright © 2013 Hung V. Trinh et al. All rights reserved. Sun, 10 Mar 2013 08:59:25 +0000 http://www.hindawi.com/journals/ijpro/2013/279590/ The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm. Si Wu, Roslyn N. Brown, Samuel H. Payne, Da Meng, Rui Zhao, Nikola Tolić, Li Cao, Anil Shukla, Matthew E. Monroe, Ronald J. Moore, Mary S. Lipton, and Ljiljana Paša-Tolić Copyright © 2013 Si Wu et al. All rights reserved. Wed, 06 Mar 2013 08:31:20 +0000 http://www.hindawi.com/journals/ijpro/2013/180605/ The accurate quantitation of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics. Mass spectrometry-based approaches have forged significant in-roads allowing accurate and sensitive quantitation and the ability to multiplex vastly complex samples through the application of robust bioinformatic tools. These relative and absolute quantitative measures using label-free, tags, or stable isotope labelling have their own strengths and limitations. The continuous development of these methods is vital for increasing reproducibility in the rapidly expanding application of quantitative proteomics in biomarker discovery and validation. This paper provides a critical overview of the primary mass spectrometry-based quantitative approaches and the current status of quantitative proteomics in biomedical research. Valerie C. Wasinger, Ming Zeng, and Yunki Yau Copyright © 2013 Valerie C. Wasinger et al. All rights reserved. Tue, 19 Feb 2013 17:23:38 +0000 http://www.hindawi.com/journals/ijpro/2013/654356/ Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%. Zhiyun Cao, Sachin Yende, John A. Kellum, and Renã A. S. Robinson Copyright © 2013 Zhiyun Cao et al. All rights reserved. Mon, 04 Feb 2013 10:11:24 +0000 http://www.hindawi.com/journals/ijpro/2013/291415/ The endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), plays an essential role in regulating the trafficking and destination of endocytosed receptors and their associated signaling molecules. Recently, we have shown that dysfunction and down-regulation of vacuolar protein sorting 4B (VPS4B), an ESCRT-III associated protein, under hypoxic conditions can lead to the abnormal accumulation of epidermal growth factor receptor (EGFR) and aberrant EGFR signaling in breast cancer. However, the pathophysiological consequences of VPS4B dysfunction remain largely elusive. In this study, we used an internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) method, which permits the direct measurement of protein synthesis, degradation and protein dynamic expression, to address the effects of VPS4B dysfunction in altering EGF-mediated protein expression. Our initial results indicate that VPS4B down-regulation decreases the expression of many proteins involved in glycolytic pathways, while increased the expression of proteins with roles in mitochondrial fatty acid β-oxidation were up-regulated in VPS4B-depleted cells. This observation is also consistent with our previous finding that hypoxia can induce VPS4B down-regulated, suggesting that the adoption of fatty acid β-oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia-mediated VPS4B dysfunction. Zhongping Liao, Stefani N. Thomas, Yunhu Wan, H. Helen Lin, David K. Ann, and Austin J. Yang Copyright © 2013 Zhongping Liao et al. All rights reserved. Sun, 03 Feb 2013 11:57:55 +0000 http://www.hindawi.com/journals/ijpro/2013/857918/ The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms. Veronica G. Anania, Daisy J. Bustos, Jennie R. Lill, Donald S. Kirkpatrick, and Laurent Coscoy Copyright © 2013 Veronica G. Anania et al. All rights reserved. Wed, 16 Jan 2013 11:57:35 +0000 http://www.hindawi.com/journals/ijpro/2013/756039/ To address the challenges associated with differential expression proteomics, label-free mass spectrometric protein quantification methods have been developed as alternatives to array-based, gel-based, and stable isotope tag or label-based approaches. In this paper, we focus on the issues associated with label-free methods that rely on quantitation based on peptide ion peak area measurement. These issues include chromatographic alignment, peptide qualification for quantitation, and normalization. In addressing these issues, we present various approaches, assembled in a recently developed label-free quantitative mass spectrometry platform, that overcome these difficulties and enable comprehensive, accurate, and reproducible protein quantitation in highly complex protein mixtures from experiments with many sample groups. As examples of the utility of this approach, we present a variety of cases where the platform was applied successfully to assess differential protein expression or abundance in body fluids, in vitro nanotoxicology models, tissue proteomics in genetic knock-in mice, and cell membrane proteomics. Xianyin Lai, Lianshui Wang, and Frank A. Witzmann Copyright © 2013 Xianyin Lai et al. All rights reserved. Tue, 15 Jan 2013 18:19:15 +0000 http://www.hindawi.com/journals/ijpro/2013/125858/ Cancer affects millions of people worldwide. Tumor mortality is substantially due to diagnosis at stages that are too late for therapies to be effective. Advances in screening methods have improved the early diagnosis, prognosis, and survival for some cancers. Several validated biomarkers are currently used to diagnose and monitor the progression of cancer, but none of them shows adequate specificity, sensitivity, and predictive value for population screening. So, there is an urgent need to isolate novel sensitive, specific biomarkers to detect the disease early and improve prognosis, especially in high-mortality tumors. Proteomic techniques are powerful tools to help in diagnosis and monitoring of treatment and progression of the disease. During the last decade, mass spectrometry has assumed a key role in most of the proteomic analyses that are focused on identifying cancer biomarkers in human serum, making it possible to identify and characterize at the molecular level many proteins or peptides differentially expressed. In this paper we summarize the results of mass spectrometry serum profiling and biomarker identification in high mortality tumors, such as ovarian, liver, lung, and pancreatic cancer. Alessandra Tessitore, Agata Gaggiano, Germana Cicciarelli, Daniela Verzella, Daria Capece, Mariafausta Fischietti, Francesca Zazzeroni, and Edoardo Alesse Copyright © 2013 Alessandra Tessitore et al. All rights reserved. Tue, 15 Jan 2013 13:26:22 +0000 http://www.hindawi.com/journals/ijpro/2013/701247/ The utility of mass spectrometry-(MS-) based proteomic platforms and their clinical applications have become an emerging field in proteomics in recent years. Owing to its selectivity and sensitivity, MS has become a key technological platform in proteomic research. Using this platform, a large number of potential biomarker candidates for specific diseases have been reported. However, due to lack of validation, none has been approved for use in clinical settings by the Food and Drug Administration (FDA). Successful candidate verification and validation will facilitate the development of potential biomarkers, leading to better strategies for disease diagnostics, prognostics, and treatment. With the recent new developments in mass spectrometers, high sensitivity, high resolution, and high mass accuracy can be achieved. This greatly enhances the capabilities of protein biomarker validation. In this paper, we describe and discuss recent developments and applications of targeted proteomics methods for biomarker validation. Jong Won Kim and Jinsam You Copyright © 2013 Jong Won Kim and Jinsam You. All rights reserved. Tue, 08 Jan 2013 11:14:17 +0000 http://www.hindawi.com/journals/ijpro/2013/219452/ Quantitative protein measurements by mass spectrometry have gained wide acceptance in research settings. However, clinical uptake of mass spectrometric protein assays has not followed suit. In part, this is due to the long-standing acceptance by regulatory agencies of immunological assays such as ELISA assays. In most cases, ELISAs provide highly accurate, sensitive, relatively inexpensive, and simple assays for many analytes. The barrier to acceptance of mass spectrometry in these situations will remain high. However, mass spectrometry provides solutions to certain protein measurements that are difficult, if not impossible, to accomplish by immunological methods. Cases where mass spectrometry can provide solutions to difficult assay development include distinguishing between very closely related protein species and monitoring biological and analytical variability due to sample handling and very high multiplexing capacity. This paper will highlight several examples where mass spectrometry has made certain protein measurements possible where immunological techniques have had a great difficulty. John E. Hale Copyright © 2013 John E. Hale. All rights reserved. Sun, 30 Dec 2012 10:49:08 +0000 http://www.hindawi.com/journals/ijpro/2012/701230/ Gary B. Smejkal Copyright © 2012 Gary B. Smejkal. All rights reserved. Sun, 25 Nov 2012 08:14:12 +0000 http://www.hindawi.com/journals/ijpro/2012/515372/ Functional proteomic profiling can help identify targets for disease diagnosis and therapy. Available methods are limited by the inability to profile many functional properties measured by enzymes kinetics. The functional proteomic profiling approach proposed here seeks to overcome such limitations. It begins with surface-based proteome separations of tissue/cell-line extracts, using SeraFILE, a proprietary protein separations platform. Enzyme kinetic properties of resulting subproteomes are then characterized, and the data integrated into proteomic profiles. As a model, SeraFILE-derived subproteomes of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine brain homogenate (BBH) and rat brain homogenate (RBH) were characterized for cAMP hydrolysis activity in the presence (challenge condition) and absence of cGMP. Functional profiles of RBH and BBH were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes. Intersample analysis showed that comparable profiles differed in only a few data points, and that distinctive subproteomes can be generated from comparable tissue samples from different animals. These results demonstrate that the proposed methods provide a means to simplify intersample differences, and to localize proteins attributable to sample-specific responses. It can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling. Amita R. Oka, Matthew P. Kuruc, Ketan M. Gujarathi, and Swapan Roy Copyright © 2012 Amita R. Oka et al. All rights reserved. Wed, 31 Oct 2012 14:26:50 +0000 http://www.hindawi.com/journals/ijpro/2012/536963/ Two-dimensional electrophoresis and mass spectrometry are undoubtedly two essential tools popularly used in proteomic analyses. Utilization of these techniques however largely depends on efficient and optimized sample preparation, regarded as one of the most crucial steps for recovering maximum amount of reliable information. The present study highlights the optimization of an effective and efficient protocol, capable of extraction of root proteins from recalcitrant phenolic rich tissues of chickpea. The widely applicable TCA-acetone and phenol-based methods have been comparatively evaluated, amongst which the latter appeared to be better suited for the sample. The phenol extraction-based method further complemented with sodium dodecyl sulphate (SDS) and pulsatory treatments proved to be the most suitable method represented by greatest spot number, good resolution, and spot intensities. All the randomly selected spots showed successful identification when subjected to further downstream MALDI-TOF and MS/MS analyses. Hence, the information obtained collectively proposes the present protein extraction protocol to be an effective one that could be applicable for recalcitrant leguminous root samples. Moniya Chatterjee, Sumanti Gupta, Anirban Bhar, and Sampa Das Copyright © 2012 Moniya Chatterjee et al. All rights reserved. Thu, 18 Oct 2012 10:06:27 +0000 http://www.hindawi.com/journals/ijpro/2012/980829/ Giardia lamblia is an “important” pathogen of humans, but as a diplomonad excavate it is evolutionarily distant from other eukaryotes and relatively little is known about its core metabolic pathways. KEGG, the widely referenced site for providing information of metabolism, does not yet include many enzymes from Giardia species. Here we identify Giardia’s core sugar metabolism using standard bioinformatic approaches. By comparing Giardia proteomes with known enzymes from other species, we have identified enzymes in the glycolysis pathway, as well as some enzymes involved in the TCA cycle and oxidative phosphorylation. However, the majority of enzymes from the latter two pathways were not identifiable, indicating the likely absence of these functionalities. We have also found enzymes from the Giardia glycolysis pathway that appear more similar to those from bacteria. Because these enzymes are different from those found in mammals, the host organisms for Giardia, we raise the possibility that these bacteria-like enzymes could be novel drug targets for treating Giardia infections. Jian Han and Lesley J. Collins Copyright © 2012 Jian Han and Lesley J. Collins. All rights reserved. Thu, 04 Oct 2012 08:31:52 +0000 http://www.hindawi.com/journals/ijpro/2012/290457/ We present a fully automated setup for performing in-line mass spectrometry (MS) analysis of conditioned media in cell cultures, in particular focusing on the peptides therein. The goal is to assess peptides secreted by cells in different culture conditions. The developed system is compatible with MS as analytical technique, as this is one of the most powerful analysis methods for peptide detection and identification. Proof of concept was achieved using the well-known mating-factor signaling in baker’s yeast, Saccharomyces cerevisiae. Our concept system holds 1 mL of cell culture medium and allows maintaining a yeast culture for, at least, 40 hours with continuous supernatant extraction (and medium replenishing). The device’s small dimensions result in reduced costs for reagents and open perspectives towards full integration on-chip. Experimental data that can be obtained are time-resolved peptide profiles in a yeast culture, including information about the appearance of mating-factor-related peptides. We emphasize that the system operates without any manual intervention or pipetting steps, which allows for an improved overall sensitivity compared to non-automated alternatives. MS data confirmed previously reported aspects of the physiology of the yeast-mating process. Moreover, matingfactor breakdown products (as well as evidence for a potentially responsible protease) were found. Emanuel Weber, Martijn W. H. Pinkse, Eda Bener-Aksam, Michael J. Vellekoop, and Peter D. E. M. Verhaert Copyright © 2012 Emanuel Weber et al. All rights reserved. Wed, 12 Sep 2012 11:52:36 +0000 http://www.hindawi.com/journals/ijpro/2012/735132/ A sequential protocol of multidimensional fractionation was optimised to enable the comparative profiling of fractions of proteomes from cultured human cells. Differential detergent fractionation was employed as a first step to obtain fractions enriched for cytosolic, membrane/organelle, nuclear, and cytoskeletal proteins. Following buffer exchange using gel-permeation chromatography, cytosolic proteins were further fractionated by 2-dimensional chromatography employing anion-exchange followed by reversed-phase steps. Chromatographic fractions were shown to be readily compatible with 1- and 2-dimensional gel electrophoresis or with direct analysis by mass spectrometry using linear-MALDI-TOF-MS. Precision of extraction was confirmed by reproducible SDS-PAGE profiles, MALDI-TOF-MS spectra, and quantitation of trypsinolytic peptides using LC-MS/MS (MRM) analyses. Solid phases were immobilised in disposable cartridges and mobile-phase flow was achieved using a combination of centrifugation and vacuum pumping. These approaches yielded parallel sample handling which was limited only by the capacities of the employed devices and which enabled both high-throughput and experimentally precise procedures, as demonstrated by the processing of experimental replicates. Protocols were employed at 10 mg scale of extracted cell protein, but these approaches would be directly applicable to both smaller and larger quantities merely by adjusting the employed solid- and mobile-phase volumes. Additional potential applications of the fractionation protocol are briefly described. David John Tooth, Varun Gopala Krishna, and Robert Layfield Copyright © 2012 David John Tooth et al. All rights reserved. Tue, 11 Sep 2012 12:14:38 +0000 http://www.hindawi.com/journals/ijpro/2012/460261/ Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL) from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC), LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome. Francesco Giorgianni, Valentina Mileo, Dominic M. Desiderio, Silvia Catinella, and Sarka Beranova-Giorgianni Copyright © 2012 Francesco Giorgianni et al. All rights reserved. Mon, 03 Sep 2012 09:25:40 +0000 http://www.hindawi.com/journals/ijpro/2012/679301/ Purpose. To utilize proteomics to discover proteins associated with significant cardiac magnetic resonance imaging (MRI) changes in marathon runners. Methods. Serum from 25 runners was analyzed by surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Proteomic profiles were compared in serum samples obtained prior to the race, at the finish line and within 7 hours after race to identify dynamic proteins correlated with cardiac MRI changes. Results. 693 protein/peptide clusters were identified using two ProteinChip surface chemistries and, of these, 116 were significantly different between the three time points. We identified 7 different patterns of protein expression change within the runners and 5 prerace protein peaks, 16 finish-line protein levels, and 15 postrace proteins which were correlated with significant postrace cardiac MRI changes. Conclusions. This study has identified baseline levels of proteins which may be predictive of risk of significant cardiac damage following a marathon race. Preliminary identification of the significant proteins suggested the involvement of cytokines and other proteins involved in stress and inflammatory response. George D. Wilson, Timothy J. Geddes, Barbara L. Pruetz, Bryan J. Thibodeau, Amy Murawka, James M. Colar, Peter A. McCullough, and Justin E. Trivax Copyright © 2012 George D. Wilson et al. All rights reserved. Thu, 16 Aug 2012 11:44:11 +0000 http://www.hindawi.com/journals/ijpro/2012/623190/ The representative tumor markers for HCC, AFP, and PIVKA-II are not satisfactory in terms of sensitivity and specificity in the early diagnosis of HCC. In search for novel markers for HCC, three-step proteome analyses were carried out in serum samples obtained from 12 patients with HCC and 10 with LC. As a first step, serum samples were subjected to antibody-based immunoaffinity column system that simultaneously removes twelve of abundant serum proteins. The concentrated flow-through was then fractionated using reversed-phase HPLC. Proteins obtained in each fraction were separated by SDS-PAGE. Serum samples obtained from patient with HCC and with LC were analyzed in parallel and their protein expression patterns were compared. A total of 83 protein bands were found to be upregulated in HCC serum. All the protein bands, the intensity of which was different between HCC and LC groups, were identified. Among them, clusterin was most significantly overexpressed (𝑃=0.023). The overexpression of serum clusterin was confirmed by ELISA using another validation set of HCC samples. Furthermore, serum clusterin was elevated in 40% of HCC cases in which both AFP and PIVKA-II were within their cut-off values. These results suggested that clusterin is a potential novel serum marker for HCC. Asako Kimura, Kazuyuki Sogawa, Mamoru Satoh, Yoshio Kodera, Osamu Yokosuka, Takeshi Tomonaga, and Fumio Nomura Copyright © 2012 Asako Kimura et al. All rights reserved. Wed, 15 Aug 2012 14:06:46 +0000 http://www.hindawi.com/journals/ijpro/2012/473843/ Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells. Amedeo Cappione III, Janet Smith, Masaharu Mabuchi, and Timothy Nadler Copyright © 2012 Amedeo Cappione et al. All rights reserved. Wed, 15 Aug 2012 10:49:16 +0000 http://www.hindawi.com/journals/ijpro/2012/641491/ Ákos Végvári, Tadashi Kondo, and John G. Marshall Copyright © 2012 Ákos Végvári et al. All rights reserved. Wed, 08 Aug 2012 10:23:17 +0000 http://www.hindawi.com/journals/ijpro/2012/342659/ Proteomic profiles of myocardial tissue in two different etiologies of heart failure were investigated using high performance liquid chromatography (HPLC)/Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Right atrial appendages from 10 patients with hemodynamically significant isolated aortic valve disease and from 10 patients with isolated symptomatic coronary heart disease were collected during elective cardiac surgery. As presented in an earlier study by our group (Baykut et al., 2006), both disease forms showed clearly different pattern distribution characteristics. Interesting enough, the classification patterns could be used for correctly sorting unknown test samples in their correct categories. However, in order to fully exploit and also validate these findings there is a definite need for unambiguous identification of the differences between different etiologies at molecular level. In this study, samples representative for the aortic valve disease and coronary heart disease were prepared, tryptically digested, and analyzed using an FT-ICR MS that allowed collision-induced dissociation (CID) of selected classifier masses. By using the fragment spectra, proteins were identified by database searches. For comparison and further validation, classifier masses were also fragmented and analyzed using HPLC-/Matrix-assisted laser desorption ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometry. Desmin and lumican precursor were examples of proteins found in aortic samples at higher abundances than in coronary samples. Similarly, adenylate kinase isoenzyme was found in coronary samples at a higher abundance. The described methodology could also be feasible in search for specific biomarkers in plasma or serum for diagnostic purposes. Jonas Bergquist, Gökhan Baykut, Maria Bergquist, Matthias Witt, Franz-Josef Mayer, and Doan Baykut Copyright © 2012 Jonas Bergquist et al. All rights reserved. Tue, 07 Aug 2012 11:24:46 +0000 http://www.hindawi.com/journals/ijpro/2012/245819/ Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC) remains disappointing with some 40–50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF) area and liver metastasis (LM) than those from main tumour body (MTB). Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined. Chandra Kirana, Hongjun Shi, Emma Laing, Kylie Hood, Rose Miller, Peter Bethwaite, John Keating, T. William Jordan, Mark Hayes, and Richard Stubbs Copyright © 2012 Chandra Kirana et al. All rights reserved. Wed, 01 Aug 2012 09:26:35 +0000 http://www.hindawi.com/journals/ijpro/2012/630409/ Chromatin-associated nonhistone proteins (CHRAPs) are readily collected from the DNaseI digested crude chromatin preparation. In this study, we show that the absolute abundance-based label-free quantitative proteomic analysis fail to identify potential CHRAPs from the CHRAP-prep. This is because that the most-highly abundant cytoplasmic proteins such as ribosomal proteins are not effectively depleted in the CHRAP-prep. Ribosomal proteins remain the top-ranked abundant proteins in the CHRAP-prep. On the other hand, we show that relative abundance-based SILAC-mediated quantitative proteomic analysis is capable of discovering the potential CHRAPs in the CHRAP-prep when compared to the whole-cell-extract. Ribosomal proteins are depleted from the top SILAC ratio-ranked proteins. In contrast, nucleus-localized proteins or potential CHRAPs are enriched in the top SILAC-ranked proteins. Consistent with this, gene-ontology analysis indicates that CHRAP-associated functions such as transcription, regulation of chromatin structures, and DNA replication and repair are significantly overrepresented in the top SILAC-ranked proteins. Some of the novel CHRAPs are confirmed using the traditional method. Notably, phenotypic assessment reveals that the top SILAC-ranked proteins exhibit the high likelihood of requirement for growth fitness under DNA damage stress. Taken together, our results indicate that the SILAC-mediated proteomic approach is capable of determining CHRAPs without prior knowledge. Han Wang, Pornpimol Tipthara, Lei Zhu, Suk Yean Poon, Kai Tang, and Jianhua Liu Copyright © 2012 Han Wang et al. All rights reserved. Mon, 30 Jul 2012 08:39:02 +0000 http://www.hindawi.com/journals/ijpro/2012/926427/ Mass spectrometry-based analyses of the low-molecular-weight fraction of serum proteome allow identifying proteome profiles (signatures) that are potentially useful in detection and classification of cancer. Several published studies have shown that multipeptide signatures selected in numerical tests have potential values for diagnostics of different types of cancer. However due to apparent problems with standardization of methodological details, both experimental and computational, none of the proposed peptide signatures analyzed directly by MALDI/SELDI-ToF spectrometry has been approved for routine diagnostics. Noteworthy, several components of proposed cancer signatures, especially those characteristic for advanced cancer, were identified as fragments of blood proteins involved in the acute phase and inflammatory response. This indicated that among cancer biomarker candidates to be possibly identified by serum proteome profiling were rather those reflecting overall influence of a disease (and the therapy) upon the human organism, than products of cancer-specific genes. Current paper focuses on changes in serum proteome that are related to response of patient’s organism to progressing malignancy and toxicity of anticancer treatment. In addition, several methodological issues that affect robustness and interlaboratory reproducibility of MS-based serum proteome profiling are discussed. Monika Pietrowska and Piotr Widłak Copyright © 2012 Monika Pietrowska and Piotr Widłak. All rights reserved. Wed, 25 Jul 2012 09:41:07 +0000 http://www.hindawi.com/journals/ijpro/2012/123076/ Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB and PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein locations for Salmonella and a framework for further investigations using computational modeling. Roslyn N. Brown, James A. Sanford, Jea H. Park, Brooke L. Deatherage, Boyd L. Champion, Richard D. Smith, Fred Heffron, and Joshua N. Adkins Copyright © 2012 Roslyn N. Brown et al. All rights reserved.