bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved wet bonding of methyl methacrylate‐tri‐n‐butylborane resin to dentin etched with ten percent phosphoric acid in the presence of ferric ions
Authors: Iwasaki Yasuhiko , Toida Tetsuya , Nakabayashi Nobuo
Published: January 2004

The objective of this study was to determine the influence of dissolved dentinal substances in demineralized dentin on the hybridization of resin for bonding to dentin. It was hypothesized that these substances, including polyelectrolytes, significantly change the substrates, which could then be assessed by the addition of Na+, Ca2+, or Fe3+ in 10% phosphoric acid. Bovine dentin specimens were etched for 10 s with a solution of 10% phosphoric acid (control) or of 22.0 mM dissolved sodium chloride (10P‐Na), calcium chloride (10P‐Ca), or ferric chloride (10P‐Fe). The specimens were then rinsed, blot‐dried, and primed three times with 5% 4‐methacryloyloxyethyl trimellitate anhydride in acetone for 60 s. Methyl methacrylate‐tri‐n‐butylborane resin was then applied. The tensile bond strength of each of the dumbbell‐shaped specimens was then measured. The fractured surfaces and modified cross‐sections were examined by scanning electron microscopy. The cross‐sections were soaked in 6N HCl for 10 s and then in 1% sodium hypochlorite for 30 min to determine the resin content in the hybridized specimens. Shrinkage of the demineralized dentins upon drying was assessed by atomic force microscopy. The tensile bond strengths were 10.8 ± 4.5 (control), 15.0 ± 7.0 (10P‐Na), 19.3 ± 5.5 (10P‐Ca), and 27.8 ± 8.1 (10P‐Fe) MPa. The atomic force microscopy studies showed that Fe3+ minimized the shrinkage by drying for 10 s but Ca2+ and Na+ did not decrease the shrinkage the same as the control. The results support the hypothesis that the monomer permeability of wet demineralized dentin is effectively improved by dissolving ferric ions in the phosphoric acid, resulting in a greater bond strength and higher resin content in the hybridized dentin. The dissolved dentinal substances, including the polyelectrolytes, had a significant influence on the characteristics of the demineralized dentin, changing the degree of hybridization and bonding. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 566–572, 2004
Effects of end boundary conditions and specimen geometry on the viscoelastic properties of cancellous bone measured by dynamic mechanical analysis
Authors: Dong X. Neil , Yeni Yener N. , Les Clifford M. , Fyhrie David P.
Published: January 2004

The viscoelastic properties of cancellous bone can be measured nondestructively in compression testing using a dynamic mechanical analyzer. In this study, we examined the effects of end boundary conditions and specimen geometry on the viscoelastic properties of cancellous bone measured by dynamic mechanical analysis. During dynamic compression testing, the cancellous bone specimens may be mechanically fixed (e.g., glued) to the loading platens or they may be free to expand across the platen surface. When specimens of cancellous bone were tested between platens with gluing, the dependence of loss tangent on frequency was not consistent with previously observed strain‐rate‐dependent mechanical behavior of cancellous bone. When long specimens of cancellous bone (length = 10 mm, diameter = 8 mm) were tested without gluing, the relationship between loss tangent and frequency depended on the level of load applied. For short specimens (length = 5 mm, diameter = 8 mm) tested without gluing, however, the frequency dependence of loss tangent agreed with existing data reported for the strain‐rate‐dependent behavior of cancellous bone and also with the frequency dependence of cortical bone viscoelasticity. Therefore, we recommend that short cancellous bone cylinders with a length of 5 mm and a diameter of 8 mm should be used without gluing in the dynamic mechanical analysis of cancellous bone. This is consistent with the American Society for Testing and Materials testing recommendations for plastics, but different from current practice for unimodal mechanical testing of cancellous bone. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 573–583, 2004
Trochophora larvae: Cell‐lineages, ciliary bands, and body regions. 1. Annelida and Mollusca
Authors: Nielsen Claus
Published: January 2004

The trochophora concept and the literature on cleavage patterns and differentiation of ectodermal structures in annelids (“polychaetes”) and molluscs are reviewed. The early development shows some variation within both phyla, and the cephalopods have a highly modified development. Nevertheless, there are conspicuous similarities between the early development of the two phyla, related to the highly conserved spiral cleavage pattern. Apical and cerebral ganglia have almost identical origin in the two phyla, and the cell‐lineage of the prototroch is identical, except for minor variations between species. The cell‐lineage of the metatrochs is almost unknown, but the telotroch of annelids and the “telotroch” of the gastropod Patella originate from the 2d‐cell, as does the gastrotroch in the few species which have been studied. The segmented annelid body, i.e. the region behind the peristome, develops through addition of new ectoderm from a ring of 2d‐cells just in front of the telotroch. This whole region is thus derived from 2d‐cells. Conversely, the mollusc body is covered by descendants of cells from both the C and D quadrants and a growth zone is not apparent. This supports the notion that the molluscs are not segmented like the annelids, and that the repeated structures seen in polyplacophorans and monplacophorans do not represent a segmentation homologous to that of the annelids. J. Exp. Zool. (Mol. Dev. Evol.) 302B: 35–68, 2004. © 2004 Wiley‐Liss, Inc.
Evo‐Devo aspects of classical and molecular data in a historical perspective
Authors: Sander Klaus , Schmidt‐Ott Urs
Published: January 2004

We discuss the interplay between evolution and development as reflected in data and concepts since about 1800. Darwin and his “continental apostle” Haeckel put the striking similarity between early vertebrate embryos in an evolutionary context. Haeckel's partly illicit generalizations discredited evolutionary thinking among early experimental embryologists who moreover noted riddles incompatible with contemporary concepts of homology and evolution. Relevant solutions were suggested by the more recent concept of ontogenetic networks that embody complex regulatory properties and genes with partly overlapping functions. Molecular data on development increasingly reveal evolutionary opportunism, for instance when a widespread signaling chain involved in primitive immune defense was apparently recruited later on for dorso‐ventral axis determination in some evolutionarily advanced insect groups. Recently, Rickettsia‐related bacteria colonizing many arthropod species were found to “manipulate” the first steps of host development to the advantage of their own propagation, but by ways that could also promote host speciation. Molecular genetics can now document evolutionary steps in ontogenetic networks. In the fruit fly for instance, the novel bicoid gene has superseded a crucial patterning function within a pre‐existing network—a case of “molecular caenogenesis.” The expression patterns of conserved genes that antagonistically determine dorso‐ventral polarity support a literal revolution envisioned almost 200 years ago. This is the dorso‐ventral inversion of the body plan in some metazoans—ascribed then to the Articulata, now to the Chordata. The final section posits that the opportunistic character of evolutionary innovations is detrimental to parsimony in development. J. Exp. Zool. (Mol. Dev. Evol.) 302B:69–91, 2004. © 2004 Wiley‐Liss, Inc.
Rupert riedl and the re‐synthesis of evolutionary and developmental biology: Body plans and evolvability
Authors: Wagner Günter P. , Laubichler Manfred D.
Published: January 2004

This paper reviews the scientific career of Rupert Riedl and his contributions to evolutionary biology. Rupert Riedl, a native of Vienna, Austria, began his career as a marine biologist who made important contributions to the systematics and anatomy of major invertebrate groups, as well as to marine ecology. When he assumed a professorship at the University of North Carolina in 1968, the predominant thinking in evolutionary biology focused on population genetics, to the virtual exclusion of most of the rest of biology. In this atmosphere Riedl developed his “systems theory” of evolution, which emphasizes the role of functional and developmental integration in limiting and enabling adaptive evolution by natural selection. The main objective of this theory is to account for the observed patterns of morphological evolution, such as the conservation of body plans. In contrast to other “alternative” theories of evolution, Riedl never denied the importance of natural selection as the driving force of evolution, but thought it necessary to contextualize natural selection with the organismal boundary conditions of adaptation. In Riedl's view development is the most important factor besides natural selection in shaping the pattern and processes of morphological evolution. J. Exp. Zool. (Mol. Dev. Evol.) 302B: 92‐104, 2004. © 2004 Wiley‐Liss, Inc.
Solid‐state glycation of β‐lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques
Authors: Fenaille François , Morgan François , Parisod Véronique , Tabet Jean‐Claude , Guy Philippe A.
Published: January 2004

The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on β‐lactoglobulin (β‐Lg) under solid‐state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS). The specific identification of lactose‐modified amino acid residues was realized using both NanoESI‐MS, NanoESI‐MS/MS (neutral loss scanning modes) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose‐induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI‐MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose‐induced modifications. Owing to the high level of β‐Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose‐modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI‐MS measurement, apart from the N‐terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75). Copyright © 2003 John Wiley & Sons, Ltd.
Qualitative study of in vivo melphalan adduct formation in the rat by miniaturized column‐switching liquid chromatography coupled with electrospray mass spectrometry
Authors: Van den Driessche Bart , Lemière Filip , Van Dongen Walter , Van der Linden Annemie , Esmans Eddy L.
Published: January 2004

In a general study of DNA adduct formation with melphalan, rats were intravenously injected with a single high dose (10 mg kg−1). Adduct formation was studied at the nucleoside level in the target organs liver, bone marrow, kidney and blood with the use of 2D liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts of dGuo and dAdo were detected under selected reaction monitoring in liver and bone marrow 10 h after administration of melphalan. In the DNA hydrolysates from kidney and blood a Gua–melphalan adduct was found, although in very low abundance. These first results of the search for in vivo‐formed melphalan adducts in the rat showed that our miniaturized LC/MS technique is useful for investigating this type of compound. More experiments will be performed in this area to gather more information about the pharmacokinetics and the quantity of adducts formed. Copyright © 2003 John Wiley & Sons, Ltd.
Measurement of the malondialdehyde–2′‐deoxyguanosine adduct in human urine by immuno‐extraction and liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry
Authors: Hoberg Anne‐Mette , Otteneder Michael , Marnett Lawrence J. , Poulsen Henrik E.
Published: January 2004

The major adduct of malondialdehyde with guanine, M1G, was measured in human urine from non‐smoking healthy individuals. M1G is a mutagenic DNA lesion and a terminal product of lipid peroxidation in vivo that may be implicated in cancer related to lifestyle and diet. On the basis of a recently developed method for the quantification of M1G as an excreted deoxynucleoside using immuno‐extraction purification, chemical NaBH4 reduction and liquid chromatography combined with atmospheric pressure chemical ionization tandem mass spectrometry, we demonstrate that the average 24 h excretion rate of M1G‐dR is about 12 ± 3.8 fmol kg−1 (n = 5). Copyright © 2004 John Wiley & Sons, Ltd.
Effects of ion‐pairing reagents on the electrospray signal suppression of sulphonated dyes and intermediates
Authors: Holčapek M. , Volná K. , Jandera P. , Kolářová L. , Lemr K. , Exner M. , Církva A.
Published: January 2004

High‐performance liquid chromatography/mass spectrometry (HPLC/MS) analysis of anionic species such as sulphonic acid dyes and intermediates requires volatile ion‐pairing mobile phase additives. Six di‐ and trialkylammonium acetates were compared with tetraalkylammonium salts and ammonium acetate in the concentration range 0–20 mmol l−1 as mobile phase additives for HPLC/MS of polysulphonated compounds. The effects of the structure and concentration of the ion‐pairing reagents on the electrospray response of mono‐, di‐ and tetrasulphonic aromatic acids and acid dyes were studied in detail. Further, five different mass analysers and instrument geometries were compared. A higher signal decrease is observed with linear geometry instruments in comparison to orthogonal or even Z‐spray geometry mass spectrometers. The concentration of mobile phase additives has a significant influence on the abundance ratios of multiply charged ions in the mass spectra of polysulphonated compounds. The competing ions of sulphonic acids may also cause significant signal suppression. Copyright © 2004 John Wiley & Sons, Ltd.
Identification of chlorinated fatty acids in fish by gas chromatography/mass spectrometry with negative ion chemical ionization of pentafluorobenzyl esters
Authors: Zhuang Wenshan , McKague A. Bruce , Reeve Douglas W. , Carey John H.
Published: January 2004

This paper reports the development of a technique for identifying and confirming chlorinated fatty acids previously detected in fish by gas chromatography (GC) with halogen‐specific detection (XSD). Fatty acid methyl esters (FAMEs) including chlorinated FAMEs within fractions of reversed‐phase high‐performance liquid chromatography of transesterified fish extracts were derivatized to pentafluorobenzyl esters, which were subjected to GC/mass spectrometry (MS) with negative ion chemical ionization (NICI). Pentafluorobenzyl esters displayed reasonably good GC characteristics, a very high ionization efficiency and a low degree of fragmentation. Chloride ion chromatograms extracted at m/z 35 and 37 from full scans were utilized for locating traces of chlorinated unknowns in the GC elution profile so that their mass spectra could be readily displayed. Significant ions displayed in the mass spectrum scanned in a narrow range of retention time where a chlorinated unknown was located were evaluated using ion chromatograms extracted at the m/z of these ions. The chromatographic peaks of those ions derived from the analyte were expected to center at that specific retention time, whereas those originating from matrix compounds were not. The isotopic patterns of chlorinated ions were also examined against their theoretical relative abundances. Using this approach, three metabolism‐related dichloro fatty acids previously identified by GC/XSD in filet extracts of white sucker sampled downstream from a bleached kraft pulp mill were confirmed: dichlorooctadecanoic, dichlorohexadecanoic and dichlorotetradecanoic acids. In addition, an isomer of dichlorotetradecanoic acid was found in a reference fish sample. As sample preparation is critical in this application, improved conditions for hydrolysis and pentafluorobenzyl esterification are also discussed Copyright © 2003 John Wiley & Sons, Ltd.
Identification and assay of wild‐type and modified nucleoside mixtures by turbo ionspray ionization tandem mass spectrometry
Authors: Benincasa Cinzia , Maiuolo Loredana , Mazzotti Fabio , Perri Enzo , Sindona Giovanni , Urso Elena
Published: January 2004

A method is presented which allows the identification and assay of a nucleoside in the presence of other analogues and homologues. The method is based on the conventional multiple reaction monitoring approach performed on the [M + H]+ ions of wild‐type and modified nucleosides produced by the turbo ionspray ionization method on a triple‐quadrupole mass spectrometer. The accuracy of the quantitative determination relies on the evaluation of a response factor ρ, which takes into account the kinetics of dissociation of the parent ions into the protonated [B + 2H]+ nucleobase ions. The evaluation of the absolute concentration of each analyte in the examined mixture does not require any previous chromatographic separation. Copyright © 2003 John Wiley & Sons, Ltd.
Structural studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix‐assisted laser desorption/ionization mass spectrometry and high‐performance liquid chromatography/electrospray ionization mass spectrometry
Authors: Cunsolo Vincenzo , Foti Salvatore , Saletti Rosaria , Gilbert Simon , Tatham Arthur S. , Shewry Peter R.
Published: January 2004

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) and reversed‐phase high‐performance liquid chromatography/electrospray ionization mass spectrometry (RP‐HPLC/ESI‐MS). For all three proteins, MALDI‐TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene‐derived amino acid sequences of the three proteins were performed by direct MALDI‐TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP‐HPLC/ESI‐MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene‐derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C‐terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post‐translational modifications of these subunits. Copyright © 2004 John Wiley & Sons, Ltd.
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

We 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

The 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

Flavonoids 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

Cytoplasmic 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

We 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

In 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

Preeclampsia 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

Uni‐ 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

Peptide 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

Part 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

Proteome 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

We 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

We 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

We 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

Toxic 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

Polycystic 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.
Identification of specific plant nucleolar phosphoproteins in a functional proteomic analysis
Authors: González‐Camacho Fernando , Medina Francisco Javier
Published: January 2004

The soluble fraction of nuclear proteins is a functionally significant fraction, since it has been shown that it contains ribonucleoproteins active in nuclear RNA metabolism. The aim of this work was to detect variations associated with cell proliferation, by comparing two‐dimensional proteomes obtained from the soluble fractions of onion nuclei isolated from actively proliferating root meristematic cells versus nonmeristematic root cells. In particular, we have studied the physicochemical features of the major nucleolar protein NopA100, a highly phosphorylated, nucleolin‐like protein. A total of 384 spots were quantified in meristematic nuclei, while only 209 were detected in nonmeristematic nuclei. The comparison of both proteomes resulted in the determination of specific spots for each proliferative state and those which were common to both cases. Furthermore, among these latter, we could discriminate quantitative differences. Interestingly, well‐known nucleolar proteins, such as RNA polymerase I, B23 and the nucleolin‐like protein NopA100, were significantly increased in proliferating cells. Western blots with anti‐NopA100 antibody demonstrated 26 spots in the meristematic sample. All the spots detected were clustered at 100 kDa and were distributed through an isoelectric point (pI) range of 4.3–6.6. In contrast, only seven spots were found in the extract from nonmeristematic nuclei, and the pI range was shortened to 4.8–6.1. These results indicate that the state of NopA100 phosphorylation correlates with the degree of nucleolar activity, i.e. the protein is more highly phosphorylated in cycling cells. We have also analyzed the bidimensional silver staining of the nucleolar organizing region (Ag‐NOR) pattern of the soluble nuclear fraction in order to identify plant cell phosphoproteins that are considered to be markers of proliferation. These experiments demonstrated that NopA100, the onion, nucleolin‐like protein, is an Ag‐NOR protein. In addition we found that the plant homologue of the vertebrate nucleolar phosphoprotein B23 migrated as two clusters of acidic spots, 43 and 42 kDa respectively in molecular mass. The differences between these features and those described for mammalian cells is discussed. Our results demonstrate that the use of protein fractionation procedures with functional significance and the location of candidate spots by indirect techniques are advantageous, complementary methods to random selection procedures for proteomic studies involving further mass spectrometry analysis.
Applicability of discovery science approach to determine biological effects of mobile phone radiation
Authors: Leszczynski Dariusz , Nylund Reetta , Joenväärä Sakari , Reivinen Jukka
Published: January 2004

We argue that the use of high‐throughput screening techniques, although expensive and laborious, is justified and necessary in studies that examine biological effects of mobile phone radiation. The “case of hsp27 protein” presented here suggests that even proteins with only modestly altered (by exposure to mobile phone radiation) expression and activity might have an impact on cell physiology. However, this short communication does not attempt to present the full scientific evidence that is far too large to be presented in a single article and that is being prepared for publication in three separate research articles. Examples of the experimental evidence presented here were designed to show the flow of experimental process demonstrating that the use of high‐throughput screening techniques might help in rapid identification of the responding proteins. This, in turn, can help in speeding up of the process of determining whether these changes might affect human health.*
The defense response of germinating maize embryos against fungal infection: A proteomics approach
Authors: Campo Sonia , Carrascal Montserrat , Coca Maria , Abián Joaquin , San Segundo Blanca
Published: January 2004

Pathogen attack on plants results in numerous host‐specific biochemical responses, the activation of some of them being critical for the ability of the plant to withstand disease. We have used high‐resolution two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry to identify proteins that are differentially expressed in response to fungal infection in maize embryos. Differential spots corresponding to induced or repressed proteins were apparent in silver stained 2‐DE gels of proteins extracted from sterile and fungal‐infected germinating embryos. Selected spots were subjected to tryptic digestion followed by identification using matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry and nanospray ion‐trap tandem mass spectrometry. Among the proteins induced in response to infection are proteins involved in protein synthesis, or in protein folding and stabilization, as well as proteins involved in oxidative stress tolerance. Additionally, the accumulation of specific pathogenesis‐related proteins in tissues of the fungal‐infected germinating embryos was studied by 2‐DE and immunoblotting.