International Journal of Proteomics The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Dimerization of Peptides by Calcium Ions: Investigation of a Calcium-Binding Motif Sun, 14 Sep 2014 00:00:00 +0000 We investigated calcium-binding motifs of peptides and their recognition of active functionalities for coordination. This investigation generates the fundamentals to design carrier material for calcium-bound peptide-peptide interactions. Interactions of different peptides with active calcium domains were investigated. Evaluation of selectivity was performed by electrospray ionization mass spectrometry by infusing solutions containing two different peptides (P1 and P2) in the presence of calcium ions. In addition to signals for monomer species, intense dimer signals are observed for the heterodimer ions ( represents the noncovalent binding of calcium with the peptide) in the positive ion mode and for ions in the negative ion mode. Monitoring of the dissociation from these mass selected dimer ions via the kinetic method provides information on the calcium affinity order of different peptide sequences. Azadeh Jamalian, Evert-Jan Sneekes, Lennard J. M. Dekker, Mario Ursem, Theo M. Luider, and Peter C. Burgers Copyright © 2014 Azadeh Jamalian et al. All rights reserved. Mapping and Identification of the Urine Proteome of Prostate Cancer Patients by 2D PAGE/MS Wed, 20 Aug 2014 06:43:39 +0000 Proteome analysis of the urine has shown that urine contains disease-specific information for a variety of urogenital system disorders, including prostate cancer (PCa). The aim of this study was to determine the protein components of urine from PCa patients. Urine from 8 patients with clinically and histologically confirmed PCa was analyzed by conventional 2D PAGE. The MS identification of the most prominent 125 spots from the urine map revealed 45 distinct proteins. According to Gene Ontology, the identified proteins are involved in a variety of biological processes, majority of them are secreted (71%), and half of them are enzymes or transporters. Comparison with the normal urine proteome revealed 11 proteins distinctive for PCa. Using Ingenuity Pathways Analysis, we have found 3 proteins (E3 ubiquitin-protein ligase rififylin, tumor protein D52, and thymidine phosphorylase) associated with cellular growth and proliferation (). The top network of functional associations between 11 proteins was Cell Death and Survival, Cell-To-Cell Signaling and Interaction, and System Development and Function . In summary, we have created an initial proteomic map of PCa patient’s urine. The results from this study provide some leads to understand the molecular bases of prostate cancer. Sanja Kiprijanovska, Sotir Stavridis, Oliver Stankov, Selim Komina, Gordana Petrusevska, Momir Polenakovic, and Katarina Davalieva Copyright © 2014 Sanja Kiprijanovska et al. All rights reserved. A Method to Determine Lysine Acetylation Stoichiometries Sun, 20 Jul 2014 09:28:50 +0000 Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodium butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions. Ernesto S. Nakayasu, Si Wu, Michael A. Sydor, Anil K. Shukla, Karl K. Weitz, Ronald J. Moore, Kim K. Hixson, Jong-Seo Kim, Vladislav A. Petyuk, Matthew E. Monroe, Ljiljiana Pasa-Tolic, Wei-Jun Qian, Richard D. Smith, Joshua N. Adkins, and Charles Ansong Copyright © 2014 Ernesto S. Nakayasu et al. All rights reserved. Prediction of Spontaneous Regression of Cervical Intraepithelial Neoplasia Lesions Grades 2 and 3 by Proteomic Analysis Sun, 15 Jun 2014 08:48:53 +0000 Regression of cervical intraepithelial neoplasia (CIN) 2-3 to CIN 1 or less is associated with immune response as demonstrated by immunohistochemistry in formaldehyde-fixed paraffin-embedded (FFPE) biopsies. Proteomic analysis of water-soluble proteins in supernatants of biopsy samples with LC-MS (LTQ-Orbitrap) was used to identify proteins predictive of CIN2-3 lesions regression. CIN2-3 in the biopsies and persistence (CIN2-3) or regression (≤CIN1) in follow-up cone biopsies was validated histologically by two experienced pathologists. In a learning set of 20 CIN2-3 (10 regressions and 10 persistence cases), supernatants were depleted of seven high abundance proteins prior to unidimensional LC-MS/MS protein analysis. Mean protein concentration was 0.81 mg/mL (range: 0.55–1.14). Multivariate statistical methods were used to identify proteins that were able to discriminate between regressive and persistent CIN2-3. The findings were validated in an independent test set of 20 CIN2-3 (10 regressions and 10 persistence cases). Multistep identification criteria identified 165 proteins. In the learning set, zinc finger protein 441 and phospholipase D6 independently discriminated between regressive and persistent CIN2-3 lesions and correctly classified all 20 patients. Nine regression and all persistence cases were correctly classified in the validation set. Zinc finger protein 441 and phospholipase D6 in supernatant samples detected by LTQ-Orbitrap can predict regression of CIN2-3. Kai-Erik Uleberg, Irene Tveiterås Øvestad, Ane Cecilie Munk, Cato Brede, Bianca van Diermen, Einar Gudlaugsson, Emiel A. M. Janssen, Anne Hjelle, and Jan P. A. Baak Copyright © 2014 Kai-Erik Uleberg et al. All rights reserved. Characterisation of the Proteome of Leptospira interrogans Serovar Canicola as a Resource for the Identification of Common Serovar Immunogenic Proteins Tue, 27 May 2014 06:22:13 +0000 Over 230 serovars of Leptospira interrogans have been identified; however few have been completely characterised. The aim of this study was to characterise the proteome of serovar Canicola and to compare this against the serovars of Copenhageni and Pomona. 2D-LC/MS analysis identified 1653 Leptospira proteins in serovar Canicola; 60 of these proteins were common to Copenhageni and Pomona, 16 of which are known to be immunogenic. This study provides the first reported proteome for serovar Canicola and suggests that proteomic comparison of different serovars could be used as a tool for identification of novel target molecules for vaccine development. P. C. Humphryes, M. E. Weeks, and N. G. Coldham Copyright © 2014 P. C. Humphryes et al. All rights reserved. Enhanced Photosynthesis and Carbon Metabolism Favor Arsenic Tolerance in Artemisia annua, a Medicinal Plant as Revealed by Homology-Based Proteomics Tue, 29 Apr 2014 00:00:00 +0000 This paper provides the first proteomic evidence of arsenic (As) tolerance and interactive regulatory network between primary and secondary metabolism in the medicinal plant, Artemisia annua. While chlorophyll fluorescence and photosynthetic rate depicted mild inhibition, there was a significant enhancement in PSI activity, whole chain, ATP, and NADPH contents in 100 μM As treatments compared to the control plants. However, a decrease in the above variables was recorded under 150 μM treatments. Proteomic decoding of the survival strategy of A. annua under As stress using 2-DE followed by MALDI-MS/MS revealed a total of 46 differentially expressed protein spots. In contrast to other plants where As inhibits photosynthesis, A. annua showed appreciable photosynthetic CO2 assimilation and allocation of carbon resources at 100 μM As concentration. While an increased accumulation of ATP synthase, ferredoxin-NADP(H) oxidoreductase, and FeS-rieske proteins supported the operation of cyclic electron transport, mdr ABC transporter protein and pcs gene might be involved in As detoxification. The most interesting observation was an increased accumulation of LEAFY like novel protein conceivably responsible for an early onset of flowering in A. annua under As stress. This study not only affirmed the role of energy metabolism proteins but also identified potential candidates responsible for As tolerance in plants. Rashmi Rai, Sarita Pandey, Alok Kumar Shrivastava, and Shashi Pandey Rai Copyright © 2014 Rashmi Rai et al. All rights reserved. In-Depth Profiling of the Peripheral Blood Mononuclear Cells Proteome for Clinical Blood Proteomics Mon, 03 Mar 2014 09:17:43 +0000 Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ. Saša Končarević, Christopher Lößner, Karsten Kuhn, Thorsten Prinz, Ian Pike, and Hans-Dieter Zucht Copyright © 2014 Saša Končarević et al. All rights reserved. Comparison of Heavy Labeled (SIL) Peptide versus SILAC Protein Internal Standards for LC-MS/MS Quantification of Hepatic Drug Transporters Tue, 25 Feb 2014 07:36:49 +0000 We studied the precision of quantification of organic anion-transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, and P-glycoprotein (P-gp) in human livers by surrogate peptide based LC-MS/MS approach using two different internal standards: stable isotope labeled peptide (SIL) versus stable isotope labeled protein (SILAC). The SIL peptides were procured commercially and the SILAC proteins were generated in-house by labeling arginine and/or lysine residues in cells expressing these transporters. Liver tissue was homogenized and the membrane fraction was isolated. The membranes were trypsin digested and the peptides were analyzed using LC-MS/MS under optimized conditions. The precision in the quantification of proteins in three independently trypsin digested samples from each liver was calculated as the standard deviation of the log transformed protein concentration. The precision of the SIL internal standard method was either slightly (, paired t-test) better than that of the SILAC method (OATP1B1, OATP1B3, and P-gp) or not different (OATP2B1). Trypsin digestion, as measured by the response of the labeled peptide derived from the SILAC protein, was consistent across liver samples. These results indicate that when maximum trypsin digestion is ensured, the SIL internal standard method can be used with confidence for quantification of drug transporters. Bhagwat Prasad and Jashvant D. Unadkat Copyright © 2014 Bhagwat Prasad and Jashvant D. Unadkat. All rights reserved. Protein-Protein Interaction Detection: Methods and Analysis Mon, 17 Feb 2014 12:30:01 +0000 Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid), TAP (tandem affinity purification), and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate the function of drug molecules. Thus, in silico methods which include sequence-based approaches, structure-based approaches, chromosome proximity, gene fusion, in silico 2 hybrid, phylogenetic tree, phylogenetic profile, and gene expression-based approaches were developed. Elucidation of protein interaction networks also contributes greatly to the analysis of signal transduction pathways. Recent developments have also led to the construction of networks having all the protein-protein interactions using computational methods for signaling pathways and protein complex identification in specific diseases. V. Srinivasa Rao, K. Srinivas, G. N. Sujini, and G. N. Sunand Kumar Copyright © 2014 V. Srinivasa Rao et al. All rights reserved. A Colorimetric Method for Monitoring Tryptic Digestion Prior to Shotgun Proteomics Mon, 10 Feb 2014 09:11:36 +0000 Tryptic digestion is an important preanalytical step in shotgun proteomics because inadequate or excessive digestion can result in a failed or incomplete experiment. Unfortunately, this step is not routinely monitored before mass spectrometry because methods available for protein digestion monitoring either are time/sample consuming or require expensive equipment. To determine if a colorimetric method (ProDM Kit) can be used to identify the extent of tryptic digestion that yields the best proteomics outcome, plasma and serum digested for 8 h and 24 h were screened with ProDM, Bioanalyzer, and LC/MS/MS, and the effect of digestion on the number of proteins identified and sequence coverage was compared. About 6% and 16% less proteins were identified when >50% of proteins were digested in plasma and serum, respectively, compared to when ~46% of proteins were digested. Average sequence coverage for albumin, haptoglobin, and serotransferrin after 2 h, 8 h, and 24 h digestion was 52%, 45%, and 45% for serum and 54%, 47%, and 42% for plasma, respectively. This paper reiterates the importance of optimizing the tryptic digestion step and demonstrates the extent to which ProDM can be used to monitor and standardize protein digestion to achieve better proteomics outcomes. Richard I. Somiari, Kutralanathan Renganathan, Stephen Russell, Steven Wolfe, Florentina Mayko, and Stella B. Somiari Copyright © 2014 Richard I. Somiari et al. All rights reserved. The Effect of Alendronate on Proteome of Hepatocellular Carcinoma Cell Lines Thu, 06 Feb 2014 12:46:59 +0000 Cancer is a life threatening disorder effecting 11 million people worldwide annually. Among various types of cancers, Hepatocellular carcinoma (HCC) has a higher rate of mortality and is the fifth leading cause of cancer related deaths around the world. Many chemotherapeutic drugs have been used for the treatment of HCC with many side effects. These drugs are inhibitors of different cell regulatory pathways. Mevalonate (MVA) pathway is an important cellular cascade vital for cell growth. A variety of inhibitors of MVA pathway have been reported for their anticancerous activity. Bisphosphonates (BPs) are members of a family involved in the treatment of skeletal complications. In recent years, their anticancer potential has been highlighted. Current study focuses on exploring the effects of alendronate (ALN), a nitrogen containing BP, on hepatocellular carcinoma cell line using genomic and proteomics approach. Our results identified ten differentially expressed proteins, of which five were up regulated and five were down regulated in ALN treated cells. Furthermore, we also performed gene expression analysis in treated and control cell lines. The study may help in understanding the molecular mechanism involved in antitumor activity of ALN, identification of possible novel drug targets, and designing new therapeutic strategies for HCC. Amber Ilyas, Zehra Hashim, Nadia Naeem, Kanwal Haneef, and Shamshad Zarina Copyright © 2014 Amber Ilyas et al. All rights reserved. Periodontal Proteomics: Wonders Never Cease! Tue, 31 Dec 2013 13:35:13 +0000 Proteins are vital parts of living organisms, as they are integral components of the physiological metabolic pathways of cells. Periodontal tissues comprise multicompartmental groups of interacting cells and matrices that provide continuous support, attachment, proprioception, and physical protection for the teeth. The proteome map, that is, complete catalogue of the matrix and cellular proteins expressed in alveolar bone, cementum, periodontal ligament, and gingiva, is to be explored for more in-depth understanding of periodontium. The ongoing research to understand the signalling pathways that allow cells to divide, differentiate, and die in controlled manner has brought us to the era of proteomics. Proteomics is defined as the study of all proteins including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, in a given cell or organism within a given environment and at a specific stage in the cell cycle. Its application to periodontal science can be used to monitor health status, disease onset, treatment response, and outcome. Proteomics can offer answers to critical, unresolved questions such as the biological basis for the heterogeneity in gingival, alveolar bone, and cemental cell populations. Harpreet Singh Grover, Shalini Kapoor, and Neha Saksena Copyright © 2013 Harpreet Singh Grover et al. All rights reserved. The Human Urinary Proteome Fingerprint Database UPdb Wed, 09 Oct 2013 15:53:15 +0000 The use of human urine as a diagnostic tool has many advantages, such as ease of sample acquisition and noninvasiveness. However, the discovery of novel biomarkers, as well as biomarker patterns, in urine is hindered mainly by a lack of comparable datasets. To fill this gap, we assembled a new urinary fingerprint database. Here, we report the establishment of a human urinary proteomic fingerprint database using urine from 200 individuals analysed by SELDI-TOF (surface enhanced laser desorption ionisation-time of flight) mass spectrometry (MS) on several chip surfaces (SEND, HP50, NP20, Q10, CM10, and IMAC30). The database currently lists 2490 unique peaks/ion species from 1172 nonredundant SELDI analyses in the mass range of 1500 to 150000. All unprocessed mass spectrometric scans are available as “.xml” data files. Additionally, 1384 peaks were included from external studies using CE (capillary electrophoresis)-MS, MALDI (matrix assisted laser desorption/ionisation), and CE-MALDI hybrids. We propose to use this platform as a global resource to share and exchange primary data derived from MS analyses in urinary research. Holger Husi, Janice B. Barr, Richard J. E. Skipworth, Nathan A. Stephens, Carolyn A. Greig, Henning Wackerhage, Rona Barron, Kenneth C. H. Fearon, and James A. Ross Copyright © 2013 Holger Husi et al. All rights reserved. Proteomic Analysis and Label-Free Quantification of the Large Clostridium difficile Toxins Tue, 27 Aug 2013 10:06:04 +0000 Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals worldwide, due to hypervirulent epidemic strains with the ability to produce increased quantities of the large toxins TcdA and TcdB. Unfortunately, accurate quantification of TcdA and TcdB from different toxinotypes using small samples has not yet been reported. In the present study, we quantify C. difficile toxins in <0.1 mL of culture filtrate by quantitative label-free mass spectrometry (MS) using data-independent analysis (MSE). In addition, analyses of both purified TcdA and TcdB as well as a standard culture filtrate were performed using gel-based and gel-independent proteomic platforms. Gel-based proteomic analysis was then used to generate basic information on toxin integrity and provided sequence confirmation. Gel-independent in-solution digestion of both toxins using five different proteolytic enzymes with MS analysis generated broad amino acid sequence coverage (91% for TcdA and 95% for TcdB). Proteomic analysis of a culture filtrate identified a total of 101 proteins, among them TcdA, TcdB, and S-layer proteins. Hercules Moura, Rebecca R. Terilli, Adrian R. Woolfitt, Yulanda M. Williamson, Glauber Wagner, Thomas A. Blake, Maria I. Solano, and John R. Barr Copyright © 2013 Hercules Moura et al. All rights reserved. Advances in Quantitative Mass Spectrometry Mon, 29 Apr 2013 11:14:52 +0000 Mu Wang, Bomie Han, and Valerie Wasinger Copyright © 2013 Mu Wang et al. All rights reserved. Quantitative Proteomics via High Resolution MS Quantification: Capabilities and Limitations Tue, 23 Apr 2013 08:36:22 +0000 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. Proteomic Analysis of the Ontogenetic Variability in Plasma Composition of Juvenile and Adult Bothrops jararaca Snakes Mon, 22 Apr 2013 12:50:22 +0000 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. Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1) and Data-Independent (MS2) Acquisitions Thu, 04 Apr 2013 09:45:29 +0000 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. iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections Mon, 11 Mar 2013 15:30:40 +0000 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. Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans Sun, 10 Mar 2013 08:59:25 +0000 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. Current Status and Advances in Quantitative Proteomic Mass Spectrometry Wed, 06 Mar 2013 08:31:20 +0000 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. Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow Tue, 19 Feb 2013 17:23:38 +0000 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. An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acid β-Oxidation in Breast Cancer Cells Mon, 04 Feb 2013 10:11:24 +0000 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. A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway Sun, 03 Feb 2013 11:57:55 +0000 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. Issues and Applications in Label-Free Quantitative Mass Spectrometry Wed, 16 Jan 2013 11:57:35 +0000 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. Serum Biomarkers Identification by Mass Spectrometry in High-Mortality Tumors Tue, 15 Jan 2013 18:19:15 +0000 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. Protein Target Quantification Decision Tree Tue, 15 Jan 2013 13:26:22 +0000 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. Advantageous Uses of Mass Spectrometry for the Quantification of Proteins Tue, 08 Jan 2013 11:14:17 +0000 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. Proteomics Sample Preparation, Preservation, and Fractionation Sun, 30 Dec 2012 10:49:08 +0000 Gary B. Smejkal Copyright © 2012 Gary B. Smejkal. All rights reserved. Functional Proteomic Profiling of Phosphodiesterases Using SeraFILE Separations Platform Sun, 25 Nov 2012 08:14:12 +0000 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.