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  • Proteomic markers of the functional sperm population in bovines 1. Comparison of low- and high-density spermatozoa following cryopreservation
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-17
    Olivier D'Amours, Gilles Frenette, Sylvie Bourassa, Ezequiel Calvo, Patrick Blondin, Robert Sullivan

    Mammalian semen contains a heterogeneous population of sperm cells. This heterogeneity results from variability in the complex processes of cell differentiation in the testis, biochemical modifications undergone by spermatozoa during transit along the male reproductive tract, interactions with secretions from accessory sex glands at ejaculation, and, in the context of reproductive technologies, in the ability of ejaculated spermatozoa to resist damage associated with freeze-thaw procedures. When submitted to density gradient centrifugation, ejaculated spermatozoa distribute themselves into two distinct populations: a low-density population characterized by low motility parameters, and a high-density population with high motility characteristics. In order to understand the origin of ejaculated spermatozoa heterogeneity, cryopreserved semen samples from bulls used by the artificial insemination (A.I.) industry were submitted to Percoll gradient centrifugation. Proteins from low and high density spermatozoa were then extracted with sodium deoxycholate and submitted to proteomic analysis using iTRAQ methodologies. Quantification of selected sperm proteins was confirmed by multiple reaction monitoring (MRM). Overall, 31 different proteins were more abundant in low-density spermatozoa while 80 different proteins were more abundant in the high-density subpopulation. Proteins enriched in high-density spermatozoa were markers of sperm functionality such as the glycolytic process, binding to the egg zona pellucida, and motility. Low-density spermatozoa are not solely characterized by loss of proteins and their associated functions. CCTs and chaperones are hallmarks of the low-density subpopulation. iTRAQ analysis revealed that other proteins such as BSPs, histone, GPX5, ELSPBP1, and CLU are overexpressed in low-density spermatozoa suggesting that these proteins represent defects occurring at different steps during the sperm journey. These differences contribute to the sperm cell heterogeneity present in mammalian semen. An accompanying paper investigates the relationship between bull fertility and sperm heterogeneity.

    更新日期:2017-11-19
  • Neuropeptidomics of the bed bug Cimex lectularius
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-17
    Reinhard Predel, Susanne Neupert, Christian Derst, Klaus Reinhardt, Christian Wegener

    The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biology. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biology of C. lectularius, neuropeptide signalling is especially interesting since it regulates feeding, diuresis, digestion as well as reproduction, and also provides potential new targets for chemical control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic analysis of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides.C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones as evidenced by the occurence of respective mass signals and immunoreactivity in neurohemal structures. Our data provides the most comprehensive peptidome of a Heteropteran species so far, and in comparison suggest that a hematophageous life style does not require qualitative adaptations of the insect peptidome.

    更新日期:2017-11-19
  • Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-16
    Jian-Ying Zhou, Lijun Chen, Bai Zhang, Yuan Tian, Tao Liu, Stefani N. Thomas, Li Chen, Michael Schnaubelt, Emily Boja, Tara Hiltke, Christopher R. Kinsinger, Henry Rodriguez, Sherri R. Davies, Shunqiang Li, Jacqueline E. Snider, Petra Erdmann-Gilmore, David L. Tabb, R. Reid Townsend, Matthew J. Ellis, Karin D. Rodland, Richard D. Smith, Steven A. Carr, Zhen Zhang, Daniel W. Chan, Hui Zhang
    更新日期:2017-11-17
  • Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down–Bottom-Up Proteomic Platform
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-16
    Tiziana Cabras, Alfredo D’Alessandro, Simone Serrao, Raffaella Isola, Federica Iavarone, Federica Vincenzoni, Giancarlo Colombo, Jörgen Ekström, Irene Messana, Massimo Castagnola
    更新日期:2017-11-17
  • TiO2 with Tandem Fractionation (TAFT): An Approach for Rapid, Deep, Reproducible, and High-Throughput Phosphoproteome Analysis
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-15
    Liangliang Ren, Chaoying Li, Wenli Shao, Weiran Lin, Fuchu He, Ying Jiang
    更新日期:2017-11-16
  • 更新日期:2017-11-15
  • Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-14
    Sharon L. Wong, Joyce To, Jerran Santos, Venkata Sita Rama Raju Allam, John P. Dalton, Steven P. Djordjevic, Sheila Donnelly, Matthew P. Padula, Maria B. Sukkar
    更新日期:2017-11-15
  • Metabolomics and Lipidomics Profiling of a Combined Mitochondrial Plus Endoplasmic Reticulum Fraction of Human Fibroblasts: A Robust Tool for Clinical Studies
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-14
    Charlotte Veyrat-Durebex, Cinzia Bocca, Stéphanie Chupin, Judith Kouassi Nzoughet, Gilles Simard, Guy Lenaers, Pascal Reynier, Hélène Blasco
    更新日期:2017-11-15
  • Improved Prediction of Bovine Leucocyte Antigens (BoLA) Presented Ligands by Use of Mass-Spectrometry-Determined Ligand and in Vitro Binding Data
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-14
    Morten Nielsen, Tim Connelley, Nicola Ternette
    更新日期:2017-11-15
  • Differential content of proteins, mRNAs, and miRNAs suggests that MDSC and their exosomes may mediate distinct immune suppressive functions
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-15
    Lucia Geis-Asteggiante, Ashton T. Belew, Virginia K. Clements, Nathan J. Edwards, Suzanne Ostrand-Rosenberg, Nagib M. El-Sayed, Catherine Fenselau

    Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the circulation and the tumor microenvironment of most cancer patients. There, MDSC suppress both adaptive and innate immunity, hindering immunotherapies. The inflammatory milieu often present in cancers facilitates MDSC suppressive activity, causing aggressive tumor progression and metastasis. MDSC from tumor-bearing mice release exosomes, which carry biologically active proteins and mediate some of the immunosuppressive functions characteristic of MDSC. Studies on other cell types have shown that exosomes may also carry RNAs which can be transferred to local and distant cells, yet the messenger RNA and microRNA cargo of MDSC-derived exosomes has not been studied to date. Here, the cargo of MDSC and their exosomes was interrogated with the goal of identifying and characterizing molecules that may facilitate MDSC suppressive potency. Because inflammation is an established driving force for MDSC suppressive activity, we used the well-established 4T1 mouse mammary carcinoma system, which includes “conventional” as well as “inflammatory” MDSC. We provide evidence that MDSC-derived exosomes carry proteins, messenger RNAs and microRNAs with different quantitative profiles than that of their parental cells. Several of these molecules have known or predicted functions consistent with MDSC suppressive activity, suggesting a potential mechanistic redundancy.

    更新日期:2017-11-15
  • Impact of Detergents on Membrane Protein Complex Isolation
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-13
    Yu-Chen Lee, Jenny Arnling Bååth, Ryan M. Bastle, Sonali Bhattacharjee, Mary Jo Cantoria, Mark Dornan, Enrique Gamero-Estevez, Lenzie Ford, Lenka Halova, Jennifer Kernan, Charlotte Kürten, Siran Li, Jerahme Martinez, Nalani Sachan, Medoune Sarr, Xiwei Shan, Nandhitha Subramanian, Keith Rivera, Darryl Pappin, Sue-Hwa Lin
    更新日期:2017-11-14
  • COMICS: Cartoon Visualization of Omics Data in Spatial Context Using Anatomical Ontologies
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-13
    Dmitrii Travin, Iaroslav Popov, Arzu Tugce Guler, Dmitry Medvedev, Suzanne van der Plas-Duivesteijn, Monica Varela, Iris C. R. M. Kolder, Annemarie H. Meijer, Herman P. Spaink, Magnus Palmblad
    更新日期:2017-11-14
  • Characterization of Proteomes Extracted through Collagen-based Stable Isotope and Radiocarbon Dating Methods
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-13
    Caroline Wadsworth, Michael Buckley
    更新日期:2017-11-14
  • Fast metabolite identification in nuclear magnetic resonance metabolomic studies: statistical peak sorting and peak overlap detection for more reliable database queries
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-14
    Pablo A. Hoijemberg, Istvan Pelczer

    A lot of time is spent by researchers in the identification of metabolites in NMR-based metabolomic studies. The usual metabolite identification starts employing public or commercial databases to match chemical shifts thought to belong to a given compound. Statistical total correlation spectroscopy (STOCSY), in use for more than a decade, speeds the process by finding statistical correlations among peaks, being able to create a better peak list as input for the database query. However, the (normally not automated) analysis becomes challenging due to the intrinsic issue of peak overlap, where correlations of more than one compound appear in the STOCSY trace. Here we present a fully automated methodology that analyzes all STOCSY traces at once (every peak is chosen as driver peak) and overcomes the peak overlap obstacle. Peak overlap detection by clustering analysis and sorting of traces (POD-CAST) first creates an overlap matrix from the STOCSY traces, then clusters the overlap traces based on their similarity and finally calculates a cumulative overlap index (COI) to account for both strong and intermediate correlations. This information is gathered in one plot to help the user identify the groups of peaks that would belong to a single molecule and perform a more reliable database query. The simultaneous examination of all traces reduces the time of analysis, compared to viewing STOCSY traces by pairs or small groups, and condenses the redundant information in the 2D STOCSY matrix into bands containing similar traces. The COI helps in the detection of overlapping peaks, which can be added to the peak list from another cross-correlated band. POD-CAST overcomes the generally overlooked and underestimated presence of overlapping peaks and it detects them to include them in the search of all compounds contributing to the peak overlap, enabling the user to accelerate the metabolite identification process with more successful database queries and searching all tentative compounds in the sample set.

    更新日期:2017-11-14
  • pSite: Amino Acid Confidence Evaluation for Quality Control of De Novo Peptide Sequencing and Modification Site Localization
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-13
    Hao Yang, Hao Chi, Wen-Jing Zhou, Wen-Feng Zeng, Chao Liu, Rui-Min Wang, Zhao-Wei Wang, Xiu-Nan Niu, Zhen-Lin Chen, Si-Min He

    MS-based de novo peptide sequencing has been improved remarkably with significant development of mass spectrometry and computational approaches, but still lacks quality control methods. Here we proposed a novel algorithm pSite to evaluate the confidence of each amino acid rather than the full-length peptides obtained by de novo peptide sequencing. A semi-supervised learning approach was used to discriminate correct amino acids from random ones and then an expectation-maximization algorithm was used to adaptively control the false amino-acid rate (FAR). On three test data sets, pSite recalled 86% more amino acids on average than PEAKS at the FAR of 5%. pSite also performed superiorly on the modification site localization problem, which is essentially a special case of amino acid confidence evaluation. On three phosphopeptide data sets, at the false localization rate of 1%, the average recall of pSite was 91% while those of Ascore and phosphoRS were 64% and 63%, respectively. pSite covered 98% of Ascore and phosphoRS results and contributed 21% more phosphorylation sites. Further analyses show that the use of distinct fragmentation features in high-resolution MS/MS spectra, such as neutral loss ions, played an important role in improving the precision of pSite. In summary, the effective and universal model together with the extensive use of spectral information makes pSite an excellent quality control tool for both de novo peptide sequencing and modification site localization.

    更新日期:2017-11-14
  • Evaluating in vitro culture medium of gut microbiome with orthogonal experimental design and metaproteomics approach
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-13
    Leyuan Li, Xu Zhang, Zhibin Ning, Janice Mayne, Jasmine I. Moore, James Butcher, Cheng-Kang Chiang, David R. Mack, Alain Stintzi, Daniel Figeys

    In vitro culture-based approaches are time- and cost-effective solutions for rapidly evaluating the effects of drugs or natural compounds against microbiomes. The nutritional composition of the culture medium is an important determinant for effectively maintaining the gut microbiome in vitro. This study combines orthogonal experimental design and a metaproteomics approach to obtain functional insights into the effects of different medium components on the microbiome. Our results show that the metaproteomic profile respond differently to medium components, including inorganic salts, bile salts, mucin and short chain fatty acids. Multi-factor analysis of variance (ANOVA) further revealed significant main and interaction effects of inorganic salts, bile salts and mucin on the different functional groups of gut microbial proteins.. While a broad regulating effect was observed on basic metabolic pathways, different medium components also showed significant modulations on cell wall/membrane/envelope biogenesis and cell motility related functions. In particular, flagellar assembly related proteins were significantly responsive to the presence of mucin. This study provides information on the functional influences of medium components on the in vitro growth of microbiome communities and gives insight on the key components that must be considered when selecting and optimizing media for culturing ex vivo microbiotas.

    更新日期:2017-11-14
  • Proteomic characterization of transcription and splicing factors associated with a metastatic phenotype in colorectal cancer
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-13
    Sofia Torres, Irene Garcia-Palmero, Consolación Marin-Vicente, Ruben A. Bartolome, Eva Calviño, María Jesús Fernandez-Aceñero, J. Ignacio Casal

    We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factors (TFs)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically-related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ. We found 240 proteins showing a significant dysregulation in highly-metastatic KM12SM cells relative to non-metastatic KM12C cells and 257 proteins in metastatic SW620 versus SW480. There were similar alterations in genuine TFs and components of the splicing machinery like UPF1, TCF7L2/TCF-4, YBX1 or SRSF3 in both cell lines. However, a significant number of alterations were cell-line specific. Functional silencing of MAFG, TFE3, TCF7L2/TCF-4 and SRSF3 in KM12 cells caused alterations in adhesion, survival, proliferation, migration and liver homing, supporting their role in metastasis. Finally, we investigated the prognostic value of the altered TFs and splicing factors in cancer patients. SRSF3 and SFPQ showed significant prognostic value. We observed that SRSF3 displayed a gradual loss of expression associated to cancer progression. Loss of SRSF3 expression was significantly associated to poor survival and shorter disease-free, particularly at early stages, in colorectal cancer.

    更新日期:2017-11-14
  • Stage Dependence, Cell-Origin Independence and Prognostic Capacity of Serum Glycan Fucosylation, β1-4 Branching, β1-6 Branching and α2-6 Sialylation in Cancer
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-12
    Shadi Ferdosi, Douglas S Rehder, Paul Maranian, Erik P. Castle, Thai H. Ho, Harvey I. Pass, Daniel W. Cramer, Karen S. Anderson, Lei Fu, David E. C. Cole, Tao Le, Xifeng Wu, Chad R. Borges

    Glycans represent a promising but only marginally accessed source of cancer markers. We previously reported the development of a molecularly bottom-up approach to plasma/serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, β1-6 branching, and core fucosylation as single analytical signals. Based on the behavior of P/S glycans established to date, we hypothesized that the alteration of P/S glycans observed in cancer would be independent of the tissue in which the tumor originated yet exhibit stage dependence that varied little between cancers classified on the basis of tumor origin. Herein, the diagnostic utility of this bottom-up approach as applied to lung cancer patients (n = 127 stage I; n = 20 stage II; n = 81 stage III; n = 90 stage IV) as well as prostate (n = 40 stage II), serous ovarian (n = 59 stage III), and pancreatic cancer patients (n = 15 rapid autopsy) compared to certifiably healthy individuals (n = 30), nominally healthy individuals (n = 166) and/or risk-matched controls (n = 300) is reported. Diagnostic performance in lung cancer was stage dependent, with markers for terminal (total) fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation most able to differentiate cases from controls. These markers behaved in a similar stage-dependent manner in other types of cancer as well. Notable differences between certifiably healthy individuals and case-matched controls were observed. These markers were not significantly elevated in liver fibrosis. Using a Cox proportional hazards regression model, the marker for α2 6 sialylation was found to predict both progression and survival in lung cancer patients after adjusting for age, gender, smoking status and stage. The potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.

    更新日期:2017-11-13
  • Identification of mature atherosclerotic plaque proteome signatures using Data-Independent Acquisition Mass Spectrometry
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-11
    Nicole Hansmeier, Josef Buttigieg, Pankaj Kumar, Shaneen Pelle, Kyoo Yoon Choi, David Kopriva, Tzu-Chiao Chao

    Atherosclerosis is a chronic inflammatory disease with complex pathobiology and one of the most common causes of cardiovascular events. The process is characterized by complex vascular remodeling processes that require the actions of numerous proteins. The composition of atherosclerotic plaque is increasingly recognized as a major factor governing the occurrence of cardiovascular or neurological symptoms. In order to gain deeper insights in the composition of atherosclerotic plaques, we created quantitative proteome profiles of advanced plaque tissues of six male patients undergoing carotid endarterectomy for stroke prevention. Using a quantitative, data-independent proteome approach, we identified 4,181 proteins with an average protein coverage of 45%. An analysis of the quantitative composition of the tissue revealed key players of vascular remodeling processes. Moreover, compared to proximal arterial tissue, 20 proteins in mature plaques were enriched, whereas 52 proteins were found in lower quantities. Among the proteins with increased abundance were prominent extracellular matrix proteins such as biglycan and lumican, whereas cytoskeletal markers for contractile smooth muscle cells (SMCs) were decreased. Taken together, this study provides the most comprehensive quantitative assessment of mature human plaque tissue to date which indicates a central role of SMCs in the structure of advanced atherosclerotic plaques.

    更新日期:2017-11-13
  • Tip-Based Fractionation of Batch-Enriched Phosphopeptides Facilitates Easy and Robust Phosphoproteome Analysis
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-10
    Alireza Dehghani, Markus Gödderz, Dominic Winter
    更新日期:2017-11-11
  • Proteomic Analyses of Cysteine Redox in High-Fat-Fed and Fasted Mouse Livers: Implications for Liver Metabolic Homeostasis
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-10
    Yixing Li, Zupeng Luo, Xilong Wu, Jun Zhu, Kai Yu, Yi Jin, Zhiwang Zhang, Shuhong Zhao, Lei Zhou
    更新日期:2017-11-11
  • Ubiquitin Conjugation Probed by Inflammation in Myeloid-Derived Suppressor Cell Extracellular Vesicles
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-10
    Katherine R. Adams, Sitara Chauhan, Divya B. Patel, Virginia K. Clements, Yan Wang, Steven M. Jay, Nathan J. Edwards, Suzanne Ostrand-Rosenberg, Catherine Fenselau
    更新日期:2017-11-11
  • Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-10
    Qin Fu, Michael P. Kowalski, Mitra Mastali, Sarah J. Parker, Kimia Sobhani, Irene van den Broek, Christie L. Hunter, Jennifer E. Van Eyk
    更新日期:2017-11-11
  • Quality Assessments of Long-term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-10
    Jian-Ying Zhou, Lijun Chen, Bai Zhang, Yuan Tian, Tao Liu, Stefani N. Thomas, Li Chen, Michael Schnaubelt, Emily Boja, Tara Hiltke, Christopher R. Kinsinger, Henry Rodriguez, Sherri R. Davies, Shunqiang Li, Jacqueline E. Snider, Petra Erdmann-Gilmore, David L. Tabb, R. Reid Townsend, Matthew J. Ellis, Karin D Rodland, Richard D. Smith, Steven A. Carr, Zhen Zhang, Daniel W. Chan, Hui Zhang

    Clinical proteomics requires large-scale analysis of human specimens to achieve statistical significance. In this study, we evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomics strategy using one channel for reference across all samples in different iTRAQ sets. A total of 148 liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses were completed, generating 6 two-dimensional (2D) LC-MS/MS datasets for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we derived a quantification confidence score based on the quality of each peptide-spectrum match (PSM) to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC-MS/MS datasets collected over a 7-month period. This study provides the first quality assessment on long-term stability and technical considerations for study design of a large-scale clinical proteomics project.

    更新日期:2017-11-11
  • Assessment of quantification precision of histone post-translational modifications by using an ion trap and down to 50,000 cells as starting material
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-09
    Qi Guo, Simone Sidoli, Benjamin A. Garcia, Xiaolu Zhao

    Histone post-translational modifications (PTMs) are fundamental players of chromatin regulation, as they contribute to editing histone chemical properties and recruiting proteins for gene transcription and DNA repair. Mass spectrometry (MS) based proteomics is currently the most widely adopted strategy for high throughput quantification of hundreds of histone PTMs. Samples such as primary tissues, complex model systems and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS. In this work, we assessed the reproducibility in quantification of histone PTMs using a wide range of starting material, i.e. from 5,000,000 to 50,000 cells. We performed the experiment using four different cell lines, i.e. HeLa, 293T, human embryonic stem cells (hESCs) and myoblasts, and we quantified a list of 205 histone peptides using ion trap MS and our in-house software. Results highlighted that the relative abundance of some histone PTMs deviated as little as just 4% when comparing high starting material with histone samples extracted from 50,000 cells, e.g. H3K9me2 (40% average abundance). Low abundance PTMs such as H3K4me2 (<3% average abundance) showed higher variability, but still around 34%. This indicates that most PTMs, and especially abundant ones, are quantified with high precision starting from low cell counts. This study will help scientists to decide whether specific experiments are feasible and to plan how much sample should be reserved for histone analysis using MS.

    更新日期:2017-11-10
  • Ultrafast Peptide Label-Free Quantification with FlashLFQ
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-08
    Robert J. Millikin, Stefan K. Solntsev, Michael R. Shortreed, Lloyd M. Smith
    更新日期:2017-11-09
  • TiO2 with Tandem Fractionation (TAFT): An Approach for Rapid, Deep, Reproducible and High-throughput Phosphoproteome Analysis
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-08
    Liangliang Ren, Chaoying Li, Wenli Shao, Weiran Lin, Fuchu He, Ying Jiang

    Mass spectrometry-based phosphoproteomic workflows traditionally require efficient prefractionation and enrichment of phosphopeptides to gain an in-depth, global and unbiased systematic investigation of phosphoproteome. Here we present TiO2 with tandem fractionation (TAFT) approach, which combines titanium dioxide (TiO2) enrichment and tandem high-pH reverse phase (HpRP) for phosphoproteome analysis in a high-throughput manner, the entire workflow takes only 3 hours to complete without laborious phosphopeptide preparation. We applied this approach to HeLa and HepG2.2.15 cells to characterize the capability of TAFT approach, which enables deep identification and quantification of more than 14,000 unique phosphopeptides in a single sample from one milligram of protein as starting materials in less than 4 hours of MS measurement. In total, we identified and quantified 21,281 phosphosites in two cell lines with >91% selectivity and high quantitative reproducibility (average Pearson correlation is 0.90 between biological replicates). More generally, the presented approach enables rapid, deep and reproducible phosphoproteome analysis in a high-throughput manner with low-cost, which should facilitate our understanding of signaling networks in a wide range of biological systems or the process of clinical applications.

    更新日期:2017-11-08
  • Improved prediction of Bovine Leucocyte Antigens (BoLA) presented ligands by use of mass spectrometry-determined ligand- and in-vitro binding data
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-08
    Morten Nielsen, Tom Connelley, Nicola Ternette

    Peptide binding to MHC class I molecules is the single most selective step in antigen presentation and the strongest single correlate to peptide cellular immunogenicity. The cost of experimentally characterizing the rules of peptide presentation for a given MHC-I molecule is extensive, and predictors of peptide-MHC interactions constitute an attractive alternative. Recently, an increasing amount of MHC presented peptides identified by mass spectrometry (MS ligands) has been published. Handling and interpretation of MS ligand data is in general challenging due to the poly-specificity nature of the data. We here outline a general pipeline for dealing with this challenge, and accurately annotate ligands to the relevant MHC-I molecule they were eluted from by use of GibbsClustering and binding motif information inferred from in-silico models. We illustrate the approach here in the context of MHC-I molecules (BoLA) of cattle. Next, we demonstrate how such annotated BoLA MS ligand data can readily be integrated with in-vitro binding affinity data in a prediction model with very high and unprecedented performance for identification of BoLA-I restricted T cell epitopes. The prediction model is freely available at http://www.cbs.dtu.dk/services/NetMHCpan/NetBoLApan

    更新日期:2017-11-08
  • Quantitative Proteomic Analysis To Identify Differentially Expressed Proteins in Myocardium of Epilepsy Using iTRAQ Coupled with Nano-LC–MS/MS
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-08
    Peng Zhang, Li Zhang, Yongguo Li, Shisheng Zhu, Minzhu Zhao, Shijia Ding, Jianbo Li
    更新日期:2017-11-08
  • Mitogen-Activated Protein Kinase Kinase 5 Regulates Proliferation and Biosynthetic Processes in Procyclic Forms of Trypanosoma brucei
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Fernanda G. Kugeratski, Michel Batista, Carla V. de Paula Lima, Lisa J. Neilson, Elizabeth Sousa da Cunha, Lyris M. de Godoy, Sara Zanivan, Marco A. Krieger, Fabricio K. Marchini
    更新日期:2017-11-08
  • SH2 Superbinder Modified Monolithic Capillary Column for the Sensitive Analysis of Protein Tyrosine Phosphorylation
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Yating Yao, Yangyang Bian, Mingming Dong, Yan Wang, Jiawen Lv, Lianfang Chen, Hongwei Wang, Jiawei Mao, Jing Dong, Mingliang Ye
    更新日期:2017-11-08
  • Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA Signaling as Prognostic Biomarker and Therapeutic Target for Colon Cancer Invasion
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Hui-Fang Hu, Wen Wen Xu, Yang Wang, Can-Can Zheng, Wei-Xia Zhang, Bin Li, Qing-Yu He
    更新日期:2017-11-08
  • Proteomic Profiling of β-hCG-Induced Spheres in BRCA1 Defective Triple Negative Breast Cancer Cells
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Satheesh Kumar Sengodan, Arathi Rajan, Sreelatha Krishnakumar Hemalatha, Revathy Nadhan, Abdul Jaleel, Priya Srinivas
    更新日期:2017-11-08
  • ADAP-GC 3.2: Graphical Software Tool for Efficient Spectral Deconvolution of Gas Chromatography–High-Resolution Mass Spectrometry Metabolomics Data
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Aleksandr Smirnov, Wei Jia, Douglas I. Walker, Dean P. Jones, Xiuxia Du
    更新日期:2017-11-08
  • Stable Isotope-Assisted Metabolic Profiling Reveals Growth Mode Dependent Differential Metabolism and Multiple Catabolic Pathways of l-Phenylalanine in Rubrivivax benzoatilyticus JA2
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Lakshmi Prasuna Mekala, Mujahid Mohammed, Sasikala Chintalapati, Venkata Ramana Chintalapati
    更新日期:2017-11-07
  • Myocardial Injury Is Distinguished from Stable Angina by a Set of Candidate Plasma Biomarkers Identified Using iTRAQ/MRM-Based Approach
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Esther Sok Hwee Cheow, Woo Chin Cheng, Terence Yap, Bamaprasad Dutta, Chuen Neng Lee, Dominique P. V. de Kleijn, Vitaly Sorokin, Siu Kwan Sze
    更新日期:2017-11-07
  • Increased Post-Translational Lysine Acetylation of Myelin Basic Protein is Associated with Peak Neurological Disability in a Mouse Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Ryan Lillico, Ting Zhou, Tina Khorshid Ahmad, Nicholas Stesco, Kiana Gozda, Jessica Truong, Jiming Kong, Ted M. Lakowski, Michael Namaka

    Citrullination of arginine residues is a post-translational modification (PTM) found on myelin basic protein (MBP), which neutralizes MBPs positive charge, and is implicated in myelin damage and multiple sclerosis (MS). Here we identify lysine acetylation as another neutralizing PTM to MBP that may be involved in myelin damage. We quantify changes in lysine and arginine PTMs on MBP derived from mice induced with an experimental autoimmune encephalomyelitis (EAE) model of MS using liquid chromatography tandem mass spectrometry. The changes in PTMs are correlated to changes in neurological disability scoring (NDS), as a marker of myelin damage. We found that lysine acetylation increased by two-fold on MBP during peak NDS post-EAE induction. We also found that, mono- and dimethyl-lysine, as well as asymmetric dimethyl-arginine residues on MBP were elevated at peak EAE disability. These findings suggest that the acetylation and methylation of lysine on MBP are PTMs associated with the neurological disability produced by EAE. Since histone deacetylase (HDAC) inhibitors have been previously shown to improve neurological disability, we also show that treatment with trichostatin A (a HDAC inhibitor) improves the NDS of EAE mice but does not change MBP acetylation.

    更新日期:2017-11-07
  • Deep learning accurately predicts estrogen receptor status in breast cancer metabolomics data
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Fadhl M. Alkawaa, Kumardeep Chaudhary, Lana X. Garmire

    Metabolomics holds the promise as a new technology to diagnose highly heterogeneous diseases. Conventionally, metabolomics data analysis for diagnosis is done using various statistical and machine learning based classification methods. However, it remains unknown if the deep neural network, a class of increasingly popular machine learning methods, is suitable to classify metabolomics data. Here we use a cohort of 271 breast cancer tissues, 204 positive estrogen receptor (ER+) and 67 negative estrogen receptor (ER-), to test the accuracies of autoencoder, a deep learning (DL) framework, as well as six widely used machine learning models, namely Random Forest (RF), Support Vector Machines (SVM), Recursive Partitioning and Regression Trees (RPART), Linear Discriminant Analysis (LDA), Prediction Analysis for Microarrays (PAM), and Generalized Boosted Models (GBM). DL framework has the highest area under the curve (AUC) of 0.93 in classifying ER+/ER- patients, compared to the other six machine learning algorithms. Furthermore, the biological interpretation of the first hidden layer reveals eight commonly enriched significant metabolomics pathways (adjusted P-value<0.05) that cannot be discovered by other machine learning methods. Among them, protein digestion & absorption and ATP-binding cassette (ABC) transporters pathways are also confirmed in integrated analysis between metabolomics and gene expression data in these samples. In summary, deep learning method shows advantages for metabolomics based breast cancer ER status classification, with both the highest prediction accuracy (AUC=0.93) and better revelation of disease biology. We encourage the adoption of autoencoder based deep learning method in the metabolomics research community for classification.

    更新日期:2017-11-07
  • Impact of detergents on membrane protein complex isolation
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Yu-Chen Lee, Jenny Arnling Bååth, Ryan M. Bastle, Sonali Bhattacharjee, Mary Jo Cantoria, Mark Dornan, Enrique Gamero-Estevez, Lenzie Ford, Lenka Halova, Jennifer Kernan, Charlotte Kürten,, Siran Li, Jerahme Martinez, Nalani Sachan, Medoune Sarr, Xiwei Shan, Nandhitha Subramanian, Keith Rivera, Darryl J. Pappin, Sue-Hwa Lin

    Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin-11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction. Further, we compared the effects of Brij 35, Triton X-100, cholate, CHAPSO, zwittergen 3-12, deoxyBIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents, except Brij35, could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that β-catenin, a known cadherin-interacting protein, was present in Cad11 immune complex among the detergents tested except Brij 35. However, the association of p120 catenin with Cad11 varied depending on the detergents used. Using isobaric tag for relative and absolute quantitation (iTRAQ) to determine the relative levels of proteins in Cad11 immune complexes, we found that DDM and Triton X-100 were more efficient than cholate in solubilization and immunoprecipitation of Cad11 and resulted in the identification of both canonical and new candidate Cad11-interacting proteins.

    更新日期:2017-11-07
  • Metabolomics and lipidomics profiling of a combined mitochondrial plus endoplasmic reticulum fraction of human fibroblasts: a robust tool for clinical studies
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-07
    Charlotte Veyrat-Durebex, Cinzia Bocca, Stéphanie Chupin, Judith Kouassi Nzoughet, Gilles Simard, Guy Lenaers, Pascal Reynier, Hélène Blasco

    Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles functions, dysfunctions and interactions. Here, we developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with an excellent reproducibility. Seventy two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12 % among which many specific of the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and “omics” combination will be useful for investigating the pathophysiology of complex diseases.

    更新日期:2017-11-07
  • MethylQuant: A Tool for Sensitive Validation of Enzyme-Mediated Protein Methylation Sites from Heavy-Methyl SILAC Data
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-03
    Aidan P. Tay, Vincent Geoghegan, Daniel Yagoub, Marc R. Wilkins, Gene Hart-Smith
    更新日期:2017-11-05
  • Proteomic Analyses of Cysteine Redox in High-fat-fed and Fasted Mouse Livers: Implications for Liver Metabolic Homeostasis
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-03
    Yixing Li, Zupeng Luo, Xilong Wu, Jun Zhu, Kai Yu, Yi Jin, Zhiwang Zhang, Shuhong Zhao, Lei Zhou

    Intensive oxidative stress occurs during high-fat-diet-induced hepatic fat deposition, suggesting a critical role for redox signaling in liver metabolism. Intriguingly, evidence shows that fasting could also result in redox-profile changes largely through reduced oxidant and/or increased antioxidant levels. However, a comprehensive landscape of redox-modified hepatic substrates is lacking, thereby hindering our understanding of liver metabolic homeostasis. In this study, we employed a proteomic approach combining iodoacetyl tandem mass tag and nano-liquid chromatography tandem mass spectrometry to quantitatively probe the effects of high-fat-feeding and fasting on in vivo redox-based cysteine modifications. Compared with control groups, ~60% of cysteine residues exhibited downregulated oxidation ratios by fasting, whereas ~94% of these ratios were upregulated by high-fat feeding. Importantly, in fasted livers, proteins exhibiting diminished cysteine oxidation were annotated in pathways associated with fatty acid metabolism, carbohydrate metabolism, insulin, peroxisome proliferator-activated receptors, oxidative respiratory chain signaling, suggesting that fasting-induced redox changes targeted major metabolic pathways and consequently resulted in hepatic lipid accumulation.

    更新日期:2017-11-03
  • PhoStar: Identifying Tandem Mass Spectra of Phosphorylated Peptides before Database Search
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-02
    Sebastian Dorl, Stephan Winkler, Karl Mechtler, Viktoria Dorfer
    更新日期:2017-11-03
  • StUbEx PLUS—A Modified Stable Tagged Ubiquitin Exchange System for Peptide Level Purification and In-Depth Mapping of Ubiquitination Sites
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-01
    Vyacheslav Akimov, Louise C. B. Olsen, Sten V. F. Hansen, Inigo Barrio-Hernandez, Michele Puglia, Søren S. Jensen, Ilia A. Solov’yov, Irina Kratchmarova, Blagoy Blagoev
    更新日期:2017-11-02
  • Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-01
    Keun Na, Heon Shin, Jin-Young Cho, Sang Hee Jung, Jaeseung Lim, Jong-Sun Lim, Eun Ah Kim, Hye Sun Kim, Ah Reum Kang, Ji Hye Kim, Jeong Min Shin, Seul-Ki Jeong, Chae-Yeon Kim, Jun Young Park, Hyung-Min Chung, Gilbert S. Omenn, William S. Hancock, Young-Ki Paik
    更新日期:2017-11-01
  • Identification and Validation of Human Missing Proteins and Peptides in Public Proteome Databases: Data Mining Strategy
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-31
    Amr Elguoshy, Yoshitoshi Hirao, Bo Xu, Suguru Saito, Ali F. Quadery, Keiko Yamamoto, Toshiaki Mitsui, Tadashi Yamamoto
    更新日期:2017-11-01
  • Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-01
    Shuiwang Hu, Luca Musante, Dorota Tataruch, Xiaomeng Xu, Oliver Kretz, Michael Henry, Paula Meleady, Haihua Luo, Hequn Zou, Yong Jiang, Harry Holthofer

    Abstract Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport and signaling. Hence membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall Protein (THP), the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral trans-membrane proteins present in human urinary vesicles. Firstly, uEVs were enriched using a “hydrostatic filtration dialysis’’ appliance (HFDa), and then the enriched uEVs and the lysates were verified by transmission electron microscopy (TEM). After using Triton X-114 phase partitioning we generated an insoluble pellet fraction (PF), aqueous phase (AP) and detergent phase (DP) -fractions and analyzed these with LC-MS/MS. Both in-gel and off-gel protein digestion methods were used to reveal increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP were much higher than of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs. Keywords

    更新日期:2017-11-01
  • Age and sex effects on plasma metabolite association networks in healthy subjects
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-01
    Alessia Vignoli, Leonardo Tenori, Claudio Luchinat, Edoardo Saccenti

    In the era of precision medicine, the analysis of simple information like sex and age can increase the potential to better diagnose and treat conditions that occur more frequently in one of the two sexes, present sex-specific symptoms and outcomes, or are characteristic of a specific age group. We present here study of the association networks constructed from an array of 22 plasma metabolites measured on a cohort of 844 healthy blood donors. Through differential network analysis we show that specific association networks can be associated with sex and age: different connectivity patterns where observed suggesting sex-related variability in several metabolic pathways (branched-chain amino acids, ketone bodies and propanoate metabolism). Reduction in metabolite hubs connectivity was also found to be associated with age in both sex groups. Network analysis was complemented with standard univariate and multivariate statistical analysis that revealed age and sex specific metabolic signatures. Our results demonstrate that the characterization of metabolite–metabolite association networks is a promising and powerful a tool to investigate the human phenotype at a molecular level.

    更新日期:2017-11-01
  • Quantitative proteomic analysis to identify differentially expressed proteins in the myocardium of epilepsy using iTRAQ coupled with nano-LC-MS/MS
    J. Proteome Res. (IF 4.268) Pub Date : 2017-11-01
    Peng Zhang, Li Zhang, Yongguo Li, Shisheng Zhu, Minzhu Zhao, Shijia Ding, Jianbo Li

    Epilepsy is a difficult-to-manage neurological disease that can result in organ damage, such as cardiac injury, that contributes to sudden unexpected death in epilepsy (SUDEP). Although recurrent seizure-induced cardiac dysregulation has been reported, the underlying molecular mechanisms are unclear. We established an epileptic model with Sprague–Dawley rats, applying isobaric tags for a relative and absolute quantification (iTRAQ)-based proteomics approach to identify differentially expressed proteins in myocardial tissue. A total of 7 proteins in the acute epilepsy group and 60 proteins in the chronic epilepsy group were identified as differentially expressed. Bioinformatics analysis suggested that the pathogenesis of cardiac injury in acute and chronic epilepsy may be due to different molecular mechanisms. Three proteins, a receptor for activated protein kinase C1 (RACK1), aldehyde dehydrogenase 6 family member A1 (ALDH6A1), and glycerol uptake/transporter 1 (Hhatl), were identified as playing crucial roles in cardiac injury during epilepsy, and were successfully confirmed by Western blot and immunohistochemistry analysis. Our study not only provides a deeper understanding of the pathophysiological mechanisms of myocardial damage in epilepsy, but also suggests some potential novel therapeutic targets for preventing cardiac injury and reducing the incidence of sudden death due to heart failure.

    更新日期:2017-11-01
  • COMICS: Cartoon Visualization of Omics Data in Spatial Context using Anatomical Ontologies
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-30
    Dmitrii Travin, Iaroslav Popov, Arzu Tugce Guler, Dmitry Medvedev, Suzanne van der Plas - Duivesteijn, Monica Varela, Iris C.R.M. Kolder, Annemarie H. Meijer, Herman P. Spaink, Magnus Palmblad

    COMICS is an interactive and open-access Web platform for integration and visualization of molecular expression data in anatomograms of zebrafish, carp and mouse model systems. Anatomical ontologies are used to map omics data across experiments and between an experiment and a particular visualization in a data dependent manner. COMICS is built on top of several existing resources. Zebrafish and mouse anatomical ontologies with their controlled vocabulary (CV) and defined hierarchy are used with the ontoCAT R package to aggregate data for comparison and visualization. Libraries from the QGIS geographical information system are used with the R packages “maps” and “maptools” to visualize and interact with molecular expression data in anatomical drawings of zebrafish and carp model systems. COMICS allows users to upload their own data from omics experiments, using any gene or protein nomenclature they wish, as long as CV terms are used to define anatomical regions or developmental stages. Common nomenclatures such as the ZFIN gene names and UniProt accessions are provided as additional support. COMICS can be used to generate publication-quality visualization of gene and protein expression across experiments. Unlike previous tools that have used anatomical ontologies to interpret imaging data in several animal models, including zebrafish, COMICS is designed to take spatially resolved data generated by dissection or fractionation and display this data in visually clear anatomical representations rather than large data tables. COMICS is optimized for ease-of-use, with a minimalistic web interface and automatic selection of the appropriate visual representation depending on the input data.

    更新日期:2017-10-31
  • We Are Not Alone: The iMOP Initiative and Its Roles in a Biology- and Disease-Driven Human Proteome Project
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-31
    Andreas Tholey, Nicolas L. Taylor, Joshua L. Heazlewood, Emøke Bendixen
    更新日期:2017-10-31
  • BioPlex Display: An Interactive Suite for Large-Scale AP–MS Protein–Protein Interaction Data
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-31
    Devin K. Schweppe, Edward L. Huttlin, J. Wade Harper, Steven P. Gygi
    更新日期:2017-10-31
  • Tip based fractionation of batch-enriched phosphopeptides facilitates easy and robust phosphoproteome analysis.
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-30
    Alireza Dehghani, Markus Gödderz, Dominic Winter

    The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation in order to reduce complexity and to allow using large amounts of starting material. For such experiments, commonly fractionation of whole cell lysate digests followed by enrichment of phosphopeptides from the single fractions is performed. We evaluated the tip-based fractionation of batch-enriched phosphopeptides as an alternative method. We compared three tip-based fractionation methods employing strong cation exchange (SCX), strong anion exchange (SAX) and C18 material for basic reversed phase (BRP) fractionation using HeLa whole cell lysate digests. We show that SCX tips are superior to BRP and SAX tips due to a more efficient retention and distribution of phosphopeptides as well as a better resolution. Furthermore, we show that tip based fractionation results in a similar performance as fractionation followed by phosphopeptide enrichment of the single fractions and outperforms analysis of unfractionated phosphopeptide enriched samples with long chromatography gradients. Our fractionation approach using SCX tips is straightforward, reproducible and requires a fraction of time, effort and instrumentation compared to the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.

    更新日期:2017-10-30
  • SH2 superbinder modified monolithic capillary column for the sensitive analysis of protein tyrosine phosphorylation
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-30
    Yating Yao, Yangyang Bian, Mingming Dong, Yan Wang, Jiawen Lv, Lianfang Chen, Hongwei Wang, Jiawei Mao, Jing Dong, Mingliang Ye

    In this study, we presents a method to specifically capture phosphotyrosine (pTyr) peptides from minute amount of sample for the sensitive analysis of protein tyrosine phosphorylation. We immobilized SH2 superbinder on a monolithic capillary column to construct a microreactor to enrich pTyr peptides. It was found the synthetic pTyr peptide could be specifically enriched by the microreactor from the peptide mixture prepared by spiking of the synthetic pTyr peptide into the tryptic digests of α-casein and β-casein with molar ratios of 1:1000:1000. The microreactor was further applied to enrich pTyr peptides from pervanadate-treated HeLa cell digests for phosphoproteomics analysis, which resulted in the identification of 796 unique pTyr sites. In contrast, the conventional SH2 superbinder-based method identified 41 pTyr sites for the same sample, only 5.2% of the number achieved by the microreactor. Finally, this microreactor was also applied to analyze the pTyr in Shc1 complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Bring together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only limited amount of sample is available.

    更新日期:2017-10-30
  • Ultrafast Peptide Label-Free Quantification with FlashLFQ
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-30
    Robert J. Millikin, Stefan K. Solntsev, Michael R. Shortreed, Lloyd M. Smith

    The rapid and accurate quantification of peptides is a critical element of modern proteomics that has become increasingly challenging as proteomic datasets grow in size and complexity. We present here FlashLFQ, a computer program for high-speed label-free quantification of peptides following a search of bottom-up mass spectrometry data. FlashLFQ is approximately an order of magnitude faster than established label-free quantification methods. The increased speed makes it practical to base quantification upon all of the charge states for a given peptide rather than solely upon the charge state that was selected for MS2 fragmentation. This increases the number of quantified peptides, improves replicate-to-replicate reproducibility, and increases quantitative accuracy. We integrated FlashLFQ into the graphical user interface of the MetaMorpheus search software, allowing it to work together with the global post-translational modification discovery (G-PTM-D) engine to accurately quantify modified peptides. FlashLFQ is also available as a NuGet package, facilitating its integration into other software, and as a standalone command line software program for the quantification of search results from other programs (e.g. MaxQuant).

    更新日期:2017-10-30
  • A highly-reproducible automated proteomics sample preparation workflow for quantitative mass spectrometry
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-30
    Qin Fu, Michael P. Kowalski, Mitra Mastali, Sarah J. Parker, Kimia Sobhani, Irene van den Broek, Christie L Hunter, Jennifer E. Van Eyk

    Sample preparation for protein quantification by mass spectrometry requires multiple processing steps including denaturation, reduction, alkylation, protease digestion, and peptide cleanup. Scaling these procedures for the analysis of numerous complex biological samples can be tedious and time-consuming, as there are many liquid transfer steps and timed reactions where technical variations can be introduced and propagated. We established an automated sample preparation workflow with a total processing time for 96 samples of 5 hours, including a 2-hour incubation with trypsin. Peptide cleanup is accomplished by online diversion during the LC/MS/MS analysis. In a selected reaction monitoring (SRM) assay targeting 6 plasma biomarkers and spiked β-galactosidase, mean intra-day and inter-day CVs for 5 serum and 5 plasma samples over 5 days were <20%. In a highly multiplexed SRM assay targeting more than 70 proteins, 90% of the transitions from 6 plasma samples repeated on 3 separate days had total CVs below 20%. Similar results were obtained when the workflow was transferred to a second site: 93% of peptides had CVs below 20%. An automated trypsin digestion workflow yields uniformly-processed samples in less than 5 hours. Reproducible quantification of peptides from more than 70 plasma proteins was observed across replicates, days, instruments, and laboratory sites, demonstrating the broad applicability of this approach.

    更新日期:2017-10-30
  • Marked differences in the submandibular salivary proteome between Sardinian alcohol-preferring and Sardinian alcohol-non preferring rats revealed by an integrated top-down/bottom-up proteomic platform.
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-30
    Tiziana Cabras, Alfredo D'Alessandro, Simone Serrao, Raffaella Isola, Federica Iavarone, Federica Vincenzoni, Giancarlo Colombo, Jorgen Ekstrom, Irene Messana, Massimo Castagnola

    Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down/bottom-up integrated approach. To this purpose submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. 200 peptides/proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. Data available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as of several unidentified peptides/proteins. sP rats expressed some proteins not detectable in sNP rats, as the glutamine/glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. Submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides, and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference/consumption of sP and sNP rats is currently unknown and requires further investigation.

    更新日期:2017-10-30
  • Microfluidic separation coupled to mass spectrometry for quantification of peanut allergens in a complex food matrix
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-30
    Rebekah L. Sayers, Lee A. Gethings, Victoria Lee, Anuradha Balasundaram, Philip J. Johnson, Justin A Marsh, Antonietta Wallace, Helen Brown, Adrian Rogers, James I. Langridge, E.N. Clare Mills

    Peanut is an important food allergen but cannot currently be reliably detected and quantified in processed foods at low levels. Three mg protein/Kg is increasingly being used as a reference dose above which precautionary allergen labeling (PAL) is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred at 0, 3, 10 or 50 mg/Kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionisation efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series performed, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimised MRM method. Whilst other peptide targets were detected with three transitions at the 50 mg/Kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could quantify reliably, and only in the chocolate dessert at 10 mg/Kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using simple extraction methodology.

    更新日期:2017-10-30
  • Why Are the Correlations between mRNA and Protein Levels so Low among the 275 Predicted Protein-Coding Genes on Human Chromosome 18?
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-27
    Ekaterina V. Poverennaya, Ekaterina V. Ilgisonis, Elena A. Ponomarenko, Arthur T. Kopylov, Victor G. Zgoda, Sergey P. Radko, Andrey V. Lisitsa, Alexander I. Archakov
    更新日期:2017-10-28
  • ADAP-GC 3.2: Graphical Software Tool for Efficient Spectral Deconvolution of Gas Chromatography-High Resolution Mass Spectrometry Metabolomics Data
    J. Proteome Res. (IF 4.268) Pub Date : 2017-10-27
    Aleksandr Smirnov, Wei Jia, Douglas I. Walker, Dean P. Jones, Xiuxia Du

    ADAP-GC is an automated computational workflow for extracting metabolite information from raw gas chromatography mass spectrometry (GC/MS) metabolomics data. Deconvolution of co-eluting analytes is a critical step in the workflow and the underlying algorithm has proven to perform similar or better than other freely available software tools. We report herein additions and improvements that have made deconvolution more efficient, user-friendly, and capable of processing high resolution GC/MS data. The improved workflow, versioned ADAP-GC 3.2, has been evaluated using both unit and high mass resolution data and the deconvolution performance has been compared to that of its predecessor. ADAP-GC 3.2 has been incorporated into MZmine 2 empowering the latter to preprocess not only LC/MS but GC/MS metabolomics data as well.

    更新日期:2017-10-27
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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