bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ion‐pair assisted recovery of matrix‐assisted laser desorption/ionization mass spectral signals from SDS‐containing peptide–protein mixtures
Authors: Rajnarayanan Rajendram V. , Wang Kuan 
Published: January 2004

AbstractWe have developed a simple and effective means of using alkylammonium ion‐pairing agents, such as cetyltetramethylammonium bromide, to recover matrix assisted laser desorption/ionization mass spectrometric (MALDI‐MS) signals from sodium dodecyl sulfate (SDS)‐containing protein and peptide samples. A two‐layer method of matrix preparation, with a bottom matrix layer of ion‐pairing agents and a top matrix layer of SDS protein samples, is essential for reproducible MALDI mass spectra with good recovery. Both buffer ions and ion‐pairing agents have profound effects on signal recovery and can be rapidly and systematically optimized. This practical technique, termed ion‐pair assisted recovery (IPAR), is compatible with major SDS‐based biotechniques and can be easily incorporated into high‐throughput proteomic analysis. Copyright © 2004 John Wiley & Sons, Ltd.
Application of a multi‐turn time‐of‐flight mass spectrometer, MULTUM II, to organic compounds ionized by matrix‐assisted laser desorption/ionization
Authors: Okumura Daisuke , Toyoda Michisato , Ishihara Morio , Katakuse Itsuo 
Published: January 2004

AbstractThe circuit shape of the ion path, or the multi‐turn, provides a solution for achieving unrestricted mass resolution from time‐of‐flight mass analyzers. The potential of a multi‐turn type mass spectrometer, the MULTUM II, with a 1.308 m circuit controlled by four toroidal electric sector fields in biological applications was examined. With matrix‐assisted laser desorption/ionization, the ion flight of 18 cycles gave a mass resolution of 10 000 for MH+ of protophorphyrin IX. This resolution was correlated with the flight length, and a resolution of 61 000 was achieved for MH+ of angiotensin I after 75 cycles or a 98.75 m total flight. The results demonstrate that the multi‐turn mass spectrometer allows not only high resolution but also very high separation of the ions of molecular species from organic compounds. Copyright © 2004 John Wiley & Sons, Ltd.
Mass spectrometry in the structural analysis of flavonoids
Authors: Cuyckens Filip , Claeys Magda 
Published: January 2004

AbstractFlavonoids are very common and widespread secondary plant metabolites. They have a wide range of biological and physiological activities and serve as chemotaxonomic marker compounds. Therefore, they have been extensively investigated both in the past and during recent years. The interest in them is still increasing. In the search for new compounds, and also in quality control, there is a need to have reliable methodology for the analysis of flavonoids. Mass spectrometry can make an invaluable contribution because of its high sensitivity, possibilities of coupling with liquid chromatography and the availability of powerful tandem mass spectrometric techniques. A review of currently available mass spectrometric methodology used in the structure elucidation of flavonoids is presented. Sample preparation, liquid chromatographic/mass spectrometric analysis and tandem mass spectrometric procedures for the characterization of flavonoid aglycones,O‐glycosides,C‐glycosides and acylated glycosides are considered. Copyright © 2004 John Wiley & Sons, Ltd.
Proteomic identification of brain proteins that interact with dynein light chain LC8
Authors: Navarro‐Lérida Inmaculada , Martínez Moreno Mónica , Roncal Fernando , Gavilanes Francisco , Albar Juan Pablo , Rodríguez‐Crespo Ignacio 
Published: January 2004

AbstractCytoplasmic dynein is a large minus end‐directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two‐hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain‐associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post‐synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.
Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) fromDrosophila melanogaster
Authors: Hernández Greco , Lalioti Vassiliki , Vandekerckhove Joel , Sierra José M. , Santarén Juan F. 
Published: January 2004

AbstractWe have identified the initiation factor 4A (eIF4A) in a two‐dimensional protein database ofDrosophilawing imaginal discs. eIF4A, a member of the DEAD‐box family of RNA helicases, forms the active eIF4F complex that in the presence of eIF4B and eIF4H unwinds the secondary structure of the 5’‐UTR of mRNAs during translational initiation. Two‐dimensional gel electrophoresis and microsequencing allowed us to purify eIF4A, and generate specific polyclonal antibodies. A combination of immunoblotting and labelling with [35S]methionine + [35S]cysteine revealed the existence of a single eIF4A isoform encoded by a previously reported gene that maps to chromosome 2L at 26A7‐9. Expression of this gene yields two mRNA species, generated by alternative splicing in the 3’‐untranslated region. The two mRNAs contain the same open reading frame and produce the identical eIF4A protein. No expression was detected of the eIF4A‐related gene CG7483. We detected eIF4A protein expression in the wing imaginal discs of severalDrosophilaspecies, and in haltere, leg 1, leg 2, leg 3, and eye‐antenna imaginal discs ofD. melanogaster. Examination of eIF4A in tumor suppressor mutants showed significantly increased (> 50%) expression in the wing imaginal discs of these larvae. We observed ubiquitous expression of eIF4A mRNA and protein duringDrosophilaembryogenesis. Yeast two‐hybrid analysis demonstrated thein vivointeraction ofDrosophilaeIF4G with theN‐terminal third of eIF4A.
Screening for N‐glycosylated proteins by liquid chromatography mass spectrometry
Authors: Bunkenborg Jakob , Pilch Bartosz J. , Podtelejnikov Alexandre V. , Wiśniewski Jacek R. 
Published: January 2004

AbstractIn the last few years mass spectrometry has become the method of choice for characterization of post‐translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N‐glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N‐glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys‐C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N‐glycosidase F and the peptides digested with trypsin before analysis by on‐line reversed‐phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N‐glycosylation sites in 77 proteins were identified.
Proteome analysis reveals elevated serum levels of clusterin in patients with preeclampsia
Authors: Watanabe Hideki , Hamada Hiromi , Yamada Naoki , Sohda Satoshi , Yamakawa‐Kobayashi Kimiko , Yoshikawa Hiroyuki , Arinami Tadao 
Published: January 2004

AbstractPreeclampsia is a pregnancy‐specific syndrome and a major cause of maternal mortality. The pathophysiology of preeclampsia is unknown, and no proteome analysis of preeclampsia has been reported. We sought to identify proteins associated with preeclampsia using a proteomic technique and performed two‐dimensional electrophoresis (2‐DE) on sera from six patients with preeclampsia and six normal pregnant women, followed by comparison of the SYPRO Ruby‐stained 2‐DE profiles. A group of overexpressed spots was identified in the limited study set. Overexpressed spots were identified as clusterin by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) followed by peptide mass fingerprinting, a protein database search, and Western blot analysis. Additionally, sera of 80 preeclamptic women and 80 normal pregnant women were processed by immunoassay methods to confirm changes in clusterin concentrations quantitatively. Immunoassays showed that clusterin levels in the 80 preeclamptic women were significantly higher than those in the 80 controls (mean ± SD; 1.62 ± 0.46 times reference level in preeclamptic women vs. 1.30 ± 0.46 times reference level in controls, P < 0.001). Proteomic analysis of serum proteins is a promising tool for studying preeclampsia pathophysiology and identifying proteins associated with preeclampsia.
Result‐driven strategies for protein identification and quantitation – a way to optimize experimental design and derive reliable results
Authors: Graber Armin , Juhasz Peter S. , Khainovski Nikita , Parker Kenneth C. , Patterson Dale H. , Martin Stephen A. 
Published: January 2004

AbstractUni‐ or multidimensional microcapillary liquid chromatography (μLC) matrix‐assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) approaches have gained significant attention for quantifying and identifying proteins in complex biological samples. The off‐line coupling of μLC with MS quantitation and MS/MS identification methods makes new result‐dependent workflows possible. A relational database is used to store the results from multiple high performance liquid chromatography runs, including information about MALDI plate positions, and both peptide and protein quantitations, and identifications. Unlike electrospray methodology, where all the decisions about which peptide to fragment, must be made during peptide fractionations, in the MALDI experiments the samples are effectively “frozen in time”. Therefore, additional MS and MS/MS spectra can be acquired, to promote more accurate quantitation or additional identifications until reliable results are derived that meet experimental design criteria. In the case of what can be designated the expression‐dependent workflow, quantitation can be detached from identification and only peak pairs with biological relevant expression changes can be selected for further MS/MS analyses. Alternatively, additional MS/MS data can be acquired to confirm tentative peptide mass fingerprint hits in what is designated a search result‐dependent workflow. In the MS data‐dependent workflow, the goal is to collect as many meaningful spectra as possible by judiciously adjusting the acquisition parameters based on characteristics of the parent masses. This level of sophistication requires the development of innovative algorithms for these three result‐dependent workflows that make MS and MS/MS analysis more efficient and also add confidence to experimental results.
A strategy to improve peptide mass fingerprinting matches through the optimization of matrix‐assisted laser desorption/ionization matrix selection and formulation
Authors: Padliya Neerav D. , Wood Troy D. 
Published: January 2004

AbstractPeptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides that result from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly‐charged ions, along with its relative insensitivity to salts and buffers, matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) is the MS method of choice for PMF. The qualitative features of a MALDI‐MS mass spectrum can be selectively tuned by varying the matrix and the solvent system used to prepare the matrix. The selective tuning of MALDI‐MS mass spectra in order to optimize PMF results is addressed in this paper. Carbonic anhydrase, hemoglobin α‐ and β‐chain, and myoglobin were digested with trypsin, and the digest was analyzed with MALDI‐MS. 2,5‐Dihydroxybenzoic acid (2,5‐DHB) and α‐cyano‐4‐hydroxycinnamic acid were prepared, using five different solvent systems: (A) 99% acetone; (B) 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA); (C) 75% ACN, 0.1% TFA; (D) formic acid:H2O: 2‐propanol (1:3:2); and (E) H2O:MeOH (2:1). Each protein was found to have a different optimum solvent system for PMF. Generally, better PMF results were obtained with 2,5‐DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest were convolved.
Application of proteome analysis to seafood authentication
Authors: Martinez Iciar , Jakobsen Friis Tone 
Published: January 2004

AbstractPart of our work aims at studying the modifications that proteins suffer in foods and use them as markers to estimate the origin and history of the product. Proteomics is a powerful approach to do this: comparison of the two‐dimensional (2‐D) maps of the intact and treated samples would permit to identify marker spots so that in the future it may be possible to estimate the treatment a foodstuff has suffered by examining its 2‐D protein map or just the selected markers. This work summarizes some of our previous studies showing the application of proteomics to the (i) identification of species and muscle tissues, (ii) characterization of post‐mortem changes in arctic and tropical species, and (iii) study of the effect of some additives during the processing of fish muscle.
Plant proteome analysis
Authors: Cánovas Francisco M. , Dumas‐Gaudot Eliane , Recorbet Ghislaine , Jorrin Jesus , Mock Hans‐Peter , Rossignol Michel 
Published: January 2004

AbstractProteome analysis is becoming a powerful tool in the functional characterization of plants. Due to the availability of vast nucleotide sequence information and based on the progress achieved in sensitive and rapid protein identification by mass spectrometry, proteome approaches open up new perspectives to analyze the complex functions of model plants and crop species at different levels. In this review, an overview is given on proteome studies performed to analyze whole plants or specific tissues with particular emphasis on important physiological processes such as germination. The chapter on subcellular proteome analysis of plants focuses on the progress achieved for plastids and mitochondria but also mentions the difficulties associated with membrane‐bound proteins of these organelles. Separate chapters are dedicated to the challenging analysis of woody plants and to the use of proteome approaches to investigate the interaction of plants with pathogens or with symbiotic organisms. Limitations of current techniques and recent conceptual and technological perspectives for plant proteomics are briefly discussed in the final chapter.
Large‐scale characterization of integral proteins from Arabidopsis vacuolar membrane by two‐dimensional liquid chromatography
Authors: Szponarski Wojciech , Sommerer Nicolas , Boyer Jean‐Christophe , Rossignol Michel , Gibrat Rémy 
Published: January 2004

AbstractWe developed a method to characterize different classes of membrane proteins within a single experiment and using simple matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) analysis. After membrane solubilization with the nondenaturing detergent n‐dodecyl‐β‐D‐maltoside, proteins were separated successively by gel filtration and ion‐exchange chromatography and finally by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). This procedure allowed to characterize 70 proteins from a membrane fraction enriched in plant vacuolar membrane (Arabidopsis), including integral proteins like the V0 complex of the H+‐ATPase, the H+‐pyrophosphatase or the glutathione S‐conjugate ATPase AtMRP1, and peripheral proteins like the subunits of the catalytic V1 complex of the H+‐ATPase. Approximately 60% of identified proteins were predicted to possess at least two trans‐membrane domains. Furthermore, proteins, with molecular masses ranging between 20 and 200 kDa were distributed into two populations with maximum frequencies at pI 5.3 and 8.9. Finally, this procedure appeared to allow the identification of proteins known to be minor in whole‐cell extracts like signaling or vesicular trafficking proteins. Almost 50% of the proteins identified were functionally unknown whereas the others confirmed that the plant vacuole is a multipurpose compartment.
Two‐dimensional reference map of Candida albicans hyphal forms
Authors: Hernández Rosa , Nombela Cesar , Diez‐Orejas Rosalía , Gil Concha 
Published: January 2004

AbstractWe have set up an in vitro model of culture of Candida albicans fungal cells that mimics the physiological conditions found in human beings in order to carry out studies of host‐pathogen interaction. Under these conditions, C. albicans undergoes dimorphic transition (yeast‐hyphae); this morphological change has been proposed as a virulence factor for this fungus. C. albicans cytoplasmic extract from hyphae cells was analyzed by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and a reference map was obtained. Protein identification was carried out by peptide mass fingerprinting or sequence tagging using a matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) or a MALDI‐TOF/TOF mass spectrometer. A total of 106 spots, excised from 2‐D gels, were analyzed. This resulted in the identification of 43 proteins involved in metabolism, 13 involved in transcription, protein synthesis, and fate, 8 involved in cell rescue, virulence, and defense, and 2 proteins of unknown function. This reference map is an important tool for future studies of protein differential expression after host‐pathogen interaction.
A technical trick for studying proteomics in parallel to transcriptomics in symbiotic root‐fungus interactions
Authors: Dumas‐Gaudot Eliane , Amiour Nardjis , Weidmann Stéphanie , Bestel‐Corre Gwénaëlle , Valot Benoit , Lenogue Sébastien , Gianinazzi‐Pearson Vivienne , Gianinazzi Silvio 
Published: January 2004

AbstractWe have developed a protocol in which proteins and mRNA can be analyzed from single root samples. This experimental design was validated in arbuscular mycorrhiza by comparing the proteins profiles obtained with those from a classical protein extraction process. It is a step forward to make simultaneous proteome and transcriptiome profiling possible.
Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies
Authors: Quero Carmen , Colomé Nuria , Prieto Maria Rosario , Carrascal Montserrat , Posada Manuel , Gelpí Emilio , Abian Joaquin 
Published: January 2004

AbstractToxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS‐affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two‐dimensional electrophoresis (2‐DE). Several problems related with serum analysis by 2‐DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty‐five of these protein spots were found to be under‐ or overexpressed in TOS patients (> three‐fold increase or decrease). Proteins in the differential spots were identified by matrix‐assisted laser desorption/ionization‐time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.
Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide
Authors: Cortón Marta , Villuendas Gemma , Botella José I. , San Millán José L. , Escobar‐Morreale Héctor F. , Peral Belén 
Published: January 2004

AbstractPolycystic ovary syndrome is a very common disease affecting 6.5% of women. It is frequently associated with obesity with the link between the two disorders being insulin resistance. From morbidly obese women submitted to surgical treatment of obesity, we obtain intra‐abdominal adipose tissue samples in order to compare protein patterns between women with and without polycystic ovary syndrome by two‐dimensional gel electrophoresis (2‐DE) analysis. Adipose tissue is a complex material extremely rich in lipids. To improve protein solubilization a lysis buffer containing 8.4 M urea, 2.4 M thiourea, 5% 3‐((3‐cholamidoproyl)dimethyl‐amino)‐1‐propanesulfonate and 50 mM dithiothreitol was used and samples were centrifuged to remove fat. Hydroxyethyl disulfide was added to increase resolution in the alkaline region (Olsson, I., Larsson, K., Palmgren, R., Bjellqvist, B., Proteomics 2002, 2, 1630–1632) and it also improved resolution in a wide pH range (3–10). Our work shows for the first time 2‐DE maps of human adipose tissue and identifies some of the proteins by mass spectrometry. This information will aid studies on metabolic diseases such as polycystic ovary syndrome, obesity, hypertension and type 2 diabetes.