The use of ubiquitin lysine mutants to characterize E2-E3 linkage specificity: Mass spectrometry offers a cautionary "tail".

Abstract:

:Oligomeric ubiquitin structures (i.e. ubiquitin "chains") may be formed through any of seven different lysine residues in the polypeptide, or via the amine group of Met 1. Different types of ubiquitin chains can confer very different biological outcomes to a protein substrate, yet the structural characteristics of E2s and E3s that determine ubiquitin linkage specificity remain poorly understood. In vitro autoubiquitylation assays combined with ubiquitin protein variants bearing individually mutated lysine residues ("K-to-R" mutants) have thus been widely used to characterize E2-E3 linkage specificity. However, how this type of assay compares to direct identification of ubiquitin linkage types using mass spectrometry (MS) has not been rigorously tested. Here, we characterize the linkage specificity of 12 different E2-E3 combinations using both approaches. The simple MS-based method described here is more robust, requires less material and is less prone to bias introduced by, e.g. the use of mutant proteins with unknown effects on E1, E2 or E3 recognition, antibodies with uncharacterized epitopes, the low dynamic range of X-ray film, and additional sources of experimental error. Indeed, our results suggest that the K-to-R assay be approached with some caution.

journal_name

Proteomics

journal_title

Proteomics

authors

Hong JH,Ng D,Srikumar T,Raught B

doi

10.1002/pmic.201500058

subject

Has Abstract

pub_date

2015-09-01 00:00:00

pages

2910-5

issue

17

eissn

1615-9853

issn

1615-9861

journal_volume

15

pub_type

杂志文章
  • Proteomics analyses of prostate cancer cells reveal cellular pathways associated with androgen resistance.

    abstract::While significant advances have been made in the diagnosis and treatment of prostate cancer, each year tens of thousands of men still die from prostate cancer in the United States. Thus, greater understanding of cellular pathways and molecular basis of prostate cancer progression in the development of androgen resista...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.201600228

    authors: Höti N,Shah P,Hu Y,Yang S,Zhang H

    更新日期:2017-03-01 00:00:00

  • Chromatographic benefits of elevated temperature for the proteomic analysis of membrane proteins.

    abstract::Integral membrane proteins (IMPs) perform crucial cellular functions and are the primary targets for most pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains and their intimate association with lipids make them difficult to handle. Numerous proteomic platforms that include LC sepa...

    journal_title:Proteomics

    pub_type: 杂志文章,评审

    doi:10.1002/pmic.200800210

    authors: Blackler AR,Speers AE,Wu CC

    更新日期:2008-10-01 00:00:00

  • Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction.

    abstract::Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TO...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200400851

    authors: Zischka H,Gloeckner CJ,Klein C,Willmann S,Swiatek-de Lange M,Ueffing M

    更新日期:2004-12-01 00:00:00

  • Meeting new challenges: The 2014 HUPO-PSI/COSMOS Workshop: 13-15 April 2014, Frankfurt, Germany.

    abstract::The Annual 2014 Spring Workshop of the Proteomics Standards Initiative (PSI) of the Human Proteome Organization (HUPO) was held this year jointly with the metabolomics COordination of Standards in MetabOlomicS (COSMOS) group. The range of existing MS standards (mzML, mzIdentML, mzQuantML, mzTab, TraML) was reviewed an...

    journal_title:Proteomics

    pub_type:

    doi:10.1002/pmic.201470164

    authors: Orchard S,Albar JP,Binz PA,Kettner C,Jones AR,Salek RM,Vizcaino JA,Deutsch EW,Hermjakob H

    更新日期:2014-11-01 00:00:00

  • Proteomic analyses of genes regulated by heterogeneous nuclear ribonucleoproteins A/B in Jurkat cells.

    abstract::Several lines of evidence suggest that hnRNPs A/B (hnRNPs A1, A2/B1, and A3) play an important role in proliferation, although the functional overlap among members of hnRNPs A/B remains largely unknown. In this study, we have employed RNAi knockdown and proteomic approaches to investigate the biological functions of h...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.201300549

    authors: Yeh YM,Chen CY,Huang PR,Hsu CW,Wu CC,Wang TC

    更新日期:2014-06-01 00:00:00

  • The stoichiometry of protein phosphorylation in adipocyte lipid droplets: analysis by N-terminal isotope tagging and enzymatic dephosphorylation.

    abstract::Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non-phosphorylated pools of proteins. ...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200800861

    authors: Kanshin E,Wang S,Ashmarina L,Fedjaev M,Nifant'ev I,Mitchell GA,Pshezhetsky AV

    更新日期:2009-11-01 00:00:00

  • Numerical approaches for quantitative analysis of two-dimensional maps: a review of commercial software and home-made systems.

    abstract::The present review attempts to cover a number of methods that have appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this review to conventional two-dimensional (2-...

    journal_title:Proteomics

    pub_type: 杂志文章,评审

    doi:10.1002/pmic.200401015

    authors: Marengo E,Robotti E,Antonucci F,Cecconi D,Campostrini N,Righetti PG

    更新日期:2005-02-01 00:00:00

  • Performance validation of an improved Xenon-arc lamp-based CCD camera system for multispectral imaging in proteomics.

    abstract::Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transillumin...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200500062

    authors: Scrivener E,Boghigian BA,Golenko E,Bogdanova A,Jackson P,Mikulskis A,Denoyer E,Courtney P,Lopez MF,Patton WF

    更新日期:2005-11-01 00:00:00

  • Sample Clean-up Strategies for ESI Mass Spectrometry Applications in Bottom-up Proteomics: Trends from 2012 to 2016.

    abstract::Bottom-up proteomics is a mass spectrometric (MS)-based approach for the characterization of peptides obtained from in-solution protein digestion. MS is favored over other methods for peptide and protein analysis because of its better sensitivity and high throughput. Inorganic ions and surfactants present in the sampl...

    journal_title:Proteomics

    pub_type: 杂志文章,评审

    doi:10.1002/pmic.201700011

    authors: Tubaon RM,Haddad PR,Quirino JP

    更新日期:2017-10-01 00:00:00

  • Multiresolution image registration for two-dimensional gel electrophoresis.

    abstract::In proteomic research, two-dimensional electrophoresis (2-D) is an important tool for investigating differential patterns of qualitative and quantitative protein expression. The strength of the technique is due to its unrivalled power of being able to separate simultaneously thousands of proteins. The key to the compa...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/1615-9861(200107)1:7<856::AID-PROT856>3.0.

    authors: Veeser S,Dunn MJ,Yang GZ

    更新日期:2001-07-01 00:00:00

  • Renal cell carcinoma: handling and treatment.

    abstract::The quality of samples and of pre-analytical steps are crucial in all biological tests, this is dramatically true in proteomics analysis. In renal cell carcinoma preparation for two-dimensional gel electrophoresis the time elapsed between sample collection and treatment, and the heterogeneity of tissues are considered...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/1615-9861(200211)2:11<1627::AID-PROT1627>3

    authors: Sarto C,Valsecchi C,Mocarelli P

    更新日期:2002-11-01 00:00:00

  • Gel-free analysis of the human brain proteome: application of liquid chromatography and mass spectrometry on biopsy and autopsy samples.

    abstract::This paper reports on the findings of the Biomedical Research Institute, as one of the participants in the pilot study of the HUPO Brain Proteome Project. A biopsy and autopsy study sample derived from human brain was distributed among the participants for proteomic analysis. In our laboratory, attention was focused o...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200600080

    authors: Dumont D,Noben JP,Verhaert P,Stinissen P,Robben J

    更新日期:2006-09-01 00:00:00

  • Peak quantification in surface-enhanced laser desorption/ionization by using mixture models.

    abstract::Surface-enhanced laser desorption/ionization (SELDI) time of flight (TOF) is a mass spectrometry technology for measuring the composition of a sampled protein mixture. A mass spectrum contains peaks corresponding to proteins in the sample. The peak areas are proportional to the measured concentrations of the correspon...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200600181

    authors: Dijkstra M,Roelofsen H,Vonk RJ,Jansen RC

    更新日期:2006-10-01 00:00:00

  • A rare and often unrecognized cerebromeningitis and hemodynamic disorder: a major cause of sudden death in somatic cell cloned piglets.

    abstract::In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, five piglets were stillborn, 22 scNT piglets died suddenly within the first week of life, and 1 piglet died after 40 days. Twelve scNT piglets are still healthy. The birth weights of compromised scNT piglets in comparison with those of normal...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200401079

    authors: Park MR,Cho SK,Lee SY,Choi YJ,Park JY,Kwon DN,Son WJ,Paik SS,Kim T,Han YM,Kim JH

    更新日期:2005-05-01 00:00:00

  • Protein expression profile of Gluconacetobacter diazotrophicus PAL5, a sugarcane endophytic plant growth-promoting bacterium.

    abstract::This is the first broad proteomic description of Gluconacetobacter diazotrophicus, an endophytic bacterium, responsible for the major fraction of the atmospheric nitrogen fixed in sugarcane in tropical regions. Proteomic coverage of G. diazotrophicus PAL5 was obtained by two independent approaches: 2-DE followed by MA...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200700912

    authors: Lery LM,Coelho A,von Kruger WM,Gonçalves MS,Santos MF,Valente RH,Santos EO,Rocha SL,Perales J,Domont GB,Teixeira KR,Bertalan M,Ferreira PC,Bisch PM

    更新日期:2008-04-01 00:00:00

  • Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry.

    abstract::This study aimed at establishing a sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using three transitio...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.201500386

    authors: Cieślak A,Kelly I,Trottier J,Verreault M,Wunsch E,Milkiewicz P,Poirier G,Droit A,Barbier O

    更新日期:2016-11-01 00:00:00

  • Popitam: towards new heuristic strategies to improve protein identification from tandem mass spectrometry data.

    abstract::In recent years, proteomics research has gained importance due to increasingly powerful techniques in protein purification, mass spectrometry and identification, and due to the development of extensive protein and DNA databases from various organisms. Nevertheless, current identification methods from spectrometric dat...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200300402

    authors: Hernandez P,Gras R,Frey J,Appel RD

    更新日期:2003-06-01 00:00:00

  • Identification and quantification of proteins differentially secreted by a pair of normal and malignant breast-cancer cell lines.

    abstract::Secreted proteins play a pivotal role in cellular functions. To better understand malignant behavior, we adapted stable isotopic labeling with amino acids in cell culture technology to identify and quantify proteins differentially released into the extracellular media by a pair of normal and malignant breast-cancer ce...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200700957

    authors: Liang X,Huuskonen J,Hajivandi M,Manzanedo R,Predki P,Amshey JR,Pope RM

    更新日期:2009-01-01 00:00:00

  • The OASIS® HLB μElution plate as a one-step platform for manual high-throughput in-gel digestion of proteins and peptide desalting.

    abstract::Separation or prefractionation of proteins by gel electrophoresis, followed by in-gel proteolytic digest and analysis by MS is a typical standard application in proteomics. For many laboratories, it is not cost-effective to use a robot with all its drawbacks. On the other hand, manual digest is time consuming and not ...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.201100354

    authors: Franz T,Li X

    更新日期:2012-08-01 00:00:00

  • A widespread picture of the Streptococcus thermophilus proteome by cell lysate fractionation and gel-based/gel-free approaches.

    abstract::Among the group of lactic acid bacteria, Streptococcus thermophilus has found a wide application in industrial processes used for the manufacture of dairy products. Taking advantage of different proteome extraction and subfractionation protocols, bacterial cytosolic and membrane proteins were isolated and resolved by ...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200601030

    authors: Salzano AM,Arena S,Renzone G,D'Ambrosio C,Rullo R,Bruschi M,Ledda L,Maglione G,Candiano G,Ferrara L,Scaloni A

    更新日期:2007-05-01 00:00:00

  • Genome-scale identification of UDP-GlcNAc-dependent pathways.

    abstract::Metabolite flux to UDP-GlcNAc and Golgi N-glycan biosynthesis regulates surface residency of glycoprotein receptors and transporters, and thus sensitivities of cells to extracellular cues. Salvage of GlcNAc increases UDP-GlcNAc and branching of N-glycans progressively, but displays an optimum for cell proliferation an...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200800208

    authors: Lau KS,Khan S,Dennis JW

    更新日期:2008-08-01 00:00:00

  • Tools for monitoring system suitability in LC MS/MS centric proteomic experiments.

    abstract::With advances in liquid chromatography coupled to tandem mass spectrometry technologies combined with the continued goals of biomarker discovery, clinical applications of established biomarkers, and integrating large multiomic datasets (i.e. "big data"), there remains an urgent need for robust tools to assess instrume...

    journal_title:Proteomics

    pub_type: 杂志文章,评审

    doi:10.1002/pmic.201400373

    authors: Bereman MS

    更新日期:2015-03-01 00:00:00

  • Fluorophore-assisted light inactivation: a high-throughput tool for direct target validation of proteins.

    abstract::To exploit advances in proteomics for drug discovery, high-throughout methods for target validation that directly address the cellular roles of proteins are required. To do this, we have characterized fluorophore-assisted light inactivation (FALI) which uses coherent or diffuse light targeted by fluorescein-labeled pr...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/1615-9861(200203)2:3<247::aid-prot247>3.0.

    authors: Beck S,Sakurai T,Eustace BK,Beste G,Schier R,Rudert F,Jay DG

    更新日期:2002-03-01 00:00:00

  • Comprehensive Peptide Analysis of Mouse Brain Striatum Identifies Novel sORF-Encoded Polypeptides.

    abstract::Bio-active peptides are involved in the regulation of most physiological processes in the body. Classical bio-active peptides (CBAPs) are cleaved from a larger precursor protein and stored in secretion vesicles from which they are released in the extracellular space. Recently, another non-classical type of bio-active ...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.201700218

    authors: Budamgunta H,Olexiouk V,Luyten W,Schildermans K,Maes E,Boonen K,Menschaert G,Baggerman G

    更新日期:2018-05-01 00:00:00

  • Ligand profiling and identification technology for searching bioactive ligands.

    abstract::We introduce a new methodology named ligand profiling and identification for effective discovery of bioactive ligands such as peptide hormones. This technology was developed from a new concept of parallel column chromatography and active fraction profiling by nano-LC MS. Traditional methods use sequential column chrom...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200500511

    authors: Baek MC,Kim SJ,Yea K,Kim Y,Lee BD,Kim J,Lee HJ,Kang MH,Choi SK,Kim JI,Lee TG,Suh PG,Ryu SH

    更新日期:2006-03-01 00:00:00

  • Protamine sulfate precipitation method depletes abundant plant seed-storage proteins: A case study on legume plants.

    abstract::Depletion of abundant proteins is one of the effective ways to improve detection and identification of low-abundance proteins. Our previous study showed that protamine sulfate precipitation (PSP) method can deplete abundant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from leaf proteins and is suitable fo...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.201400488

    authors: Kim YJ,Wang Y,Gupta R,Kim SW,Min CW,Kim YC,Park KH,Agrawal GK,Rakwal R,Choung MG,Kang KY,Kim ST

    更新日期:2015-05-01 00:00:00

  • jTraqX: a free, platform independent tool for isobaric tag quantitation at the protein level.

    abstract::Many proteomic studies focus on quantitative aspects, using different stable isotope labeling techniques that require specialized software to analyze the generated data. Here we present jTraqX, an easy-to-use tool for processing and visualizing protein quantification data. jTraqX is platform independent and is compati...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200900374

    authors: Muth T,Keller D,Puetz SM,Martens L,Sickmann A,Boehm AM

    更新日期:2010-03-01 00:00:00

  • Extensive characterization of peptides from Panax ginseng C. A. Meyer using mass spectrometric approach.

    abstract::Panax ginseng is an important herb that has clear effects on the treatment of diverse diseases. Until now, the natural peptide constitution of this herb remains unclear. Here, we conduct an extensive characterization of Ginseng peptidome using MS-based data mining and sequencing. The screen on the charge states of pre...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.201600183

    authors: Ye X,Zhao N,Yu X,Han X,Gao H,Zhang X

    更新日期:2016-11-01 00:00:00

  • Comparative glycoproteomics based on lectins affinity capture of N-linked glycoproteins from human Chang liver cells and MHCC97-H cells.

    abstract::We present here an effective technique for the large-scale separation and identification of N-linked glycoproteins from Chang liver cells, the human normal liver cells. To enrich N-linked glycoproteins from the whole cells, a procedure containing the lysis of human liver cells, the solubilization of total proteins, le...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200600041

    authors: Xu Z,Zhou X,Lu H,Wu N,Zhao H,Zhang L,Zhang W,Liang YL,Wang L,Liu Y,Yang P,Zha X

    更新日期:2007-07-01 00:00:00

  • Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger.

    abstract::A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilize...

    journal_title:Proteomics

    pub_type: 杂志文章

    doi:10.1002/pmic.200600219

    authors: White MY,Tchen AS,McCarron HC,Hambly BD,Jeremy RW,Cordwell SJ

    更新日期:2006-12-01 00:00:00