bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplex proteomic analysis by two‐dimensional differential in‐gel electrophoresis
Authors: Knowles Michael R. , Cervino Sandra , Skynner Heather A. , Hunt Stephen P. , de Felipe Carmen , Salim Kamran , Meneses‐Lorente Georgina , McAllister George , Guest Paul C. 
Published: July 2003

AbstractThis paper describes the use of fluorescence two‐dimensional differential in‐gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK1R−/−) and wild type (NK1R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK1R−/− and NK1R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two‐dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In‐gel Analysis software. A Cy2‐labeled sample pool was co‐electrophoresed with all Cy3‐ and Cy5‐labeled sample pairs as an internal standard providing a link for inter‐gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK1R−/− mice compared to NK1R+/+ controls. Matrix assisted laser desorption/ionisation‐time of flight (MALDI‐TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.
A sensitive two‐color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels
Authors: Jing Debra , Agnew Jakyoung , Patton Wayne F. , Hendrickson Jill , Beechem Joseph M. 
Published: July 2003

AbstractDNA‐binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein‐nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous 32P‐labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility‐shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR® Green EMSA dye, while proteins are subsequently detected with SYPRO® Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel‐shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein‐nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.
Evaluation of saturation labelling two‐dimensional difference gel electrophoresis fluorescent dyes
Authors: Shaw Joanne , Rowlinson Rachel , Nickson Janice , Stone Tim , Sweet Alison , Williams Karen , Tonge Robert 
Published: July 2003

AbstractTwo‐dimensional difference gel electrophoresis (2‐D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye™ DIGE Cy™3 and Cy5 dyes, which aim to overcome these limitations through saturation‐labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation‐labelled gels for MS analysis. The saturation‐labelling dyes were superior in sensitivity to their minimal‐labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2‐D spot pattern was significantly altered from that of unlabelled or minimal‐labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation‐labelled gels eliminating the need for post‐stained preparative gels.
An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence‐based protein detection technologies
Authors: Schulenberg Birte , Arnold Brad , Patton Wayne F. 
Published: July 2003

AbstractUnfortunately, conventional large‐format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large‐format two‐dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide‐based matrix has been developed that, upon polymerization, facilitates the handling of one‐dimensional and two‐dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer‐reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high‐molecular‐weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro‐Q Diamond phosphoprotein stain, Pro‐Q Emerald glycoprotein stain and SYPRO® Ruby protein gel stain, respectively.
Two‐dimensional electrophoresis replica blotting: A valuable technique for the immunological and biochemical characterization of single components of complex extracts
Authors: Petersen Arnd 
Published: July 2003

AbstractThe combination of the high resolution electrophoresis (2‐DE) with subsequent transfer onto a protein‐binding membrane (blotting), immunological detection, and/or N‐terminal sequencing is a powerful tool to identify and characterize single components of complex protein mixtures. Direct comparison of protein staining, immunological detection, and biochemical characterization of single protein spots was achieved by the replica blotting technique. The proteins were transferred from one two‐dimensional gel onto several blotting membranes one after another. A canon of methods has been employed to identify and characterize allergens from different allergen sources. We have studied single major allergens as well as related major allergens from different grass species (“allergen groups”) using patients’ sera and allergen‐specific monoclonal antibodies. The biochemical structure of the allergenic components has been analyzed by N‐terminal and internal protein sequencing, precise mass determinations by matrix‐assisted laser desorption/ionization mass spectrometry and investigations on post‐translational modifications such as glycosylation. Here, we give a general survey of methods, and we describe an array of techniques suitable for characterization and identification of components of complex extracts, even if there is little or no previous information available.
Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine‐reactive dye in combination with alkaline phosphatase‐ and horseradish peroxidase‐antibody conjugates
Authors: Martin Karen , Hart Courtenay , Liu Jixiang , Leung Wai‐Yee , Patton Wayne F. 
Published: July 2003

AbstractThree‐color fluorescence detection methods are described based upon covalently coupling the dye 2‐methoxy‐2,4‐diphenyl‐2(2H)‐furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline‐phosphatase‐conjugated reporter molecules in combination with the fluorogenic substrate 9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one‐7‐yl) phosphate (DDAO‐phosphate) as well as horseradish peroxidase‐conjugated reporter molecules in combination with the new fluorogenic substrate Amplex® Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi‐illuminators combined with photographic or charge‐coupled device (CCD) cameras, and xenon‐arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser‐based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.
Protein microarrays using liquid phase fractionation of cell lysates
Authors: Yan Fang , Sreekumar Arun , Laxman Bharathi , Chinnaiyan Arul M. , Lubman David M. , Barder Timothy J. 
Published: July 2003

AbstractWe describe an approach in which protein microarrays are produced using a two‐dimensional (2‐D) liquid phase fractionation of cell lysates. The method involves a pI‐based fractionation using chromatofocusing in the first dimension followed by nonporous reversed‐phase high‐performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero‐diagnostic markers for prostate cancer. Importantly, this method allows for use of post‐translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.
In vitro protein microarrays for detecting protein‐protein interactions: Application of a new method for fluorescence labeling of proteins
Authors: Kawahashi Yuko , Doi Nobuhide , Takashima Hideaki , Tsuda Chizuru , Oishi Yuko , Oyama Rieko , Yonezawa Masato , Miyamoto‐Sato Etsuko , Yanagawa Hiroshi 
Published: July 2003

AbstractProtein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein‐protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell‐free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore‐puromycin conjugate was incorporated into a protein at the C‐terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein‐protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high‐throughput format and also combined with in vitro display technologies such as the streptavidin‐biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227–230), our method should be useful for large‐scale analysis of protein‐protein interactions.
Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye
Authors: Martin Karen , Steinberg Thomas H. , Cooley Laurie A. , Gee Kyle R. , Beechem Joseph M. , Patton Wayne F. 
Published: July 2003

AbstractUltrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post‐translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((γ‐32P)ATP(γ‐33P)ATP labeling) or phosphoamino acid‐selective antibodies. Both of these methods suffer from relatively well‐known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro‐Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro‐Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid‐selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312–625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro‐Q Diamond dye readily permits differentiation between specific and non‐specific peptide labeling at picogram to subpicogram levels of detection sensitivity.
Profiling the progression of cancer: Separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis
Authors: O’Neil Kimberly A. , Miller Fred R. , Barder Timothy J. , Lubman David M. 
Published: July 2003

AbstractThe heterogeneity of cellular protein expression has stimulated development of separations targeting smaller groups of related proteins rather than entire proteomes. The following work describes the development of a technique for the characterization of membrane subproteomes from five different breast epithelial cell lines. Intact membrane proteins are separated by hydrophobicity in the first dimension using nonporous reversed‐phase high‐performance liquid chromatography (RP‐HPLC) to generate unique chromatographic profiles. Fractions of eluent are further separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to create distinct banding patterns. This hybrid liquid phase/gel phase method circumvents issues of membrane protein precipitation and provides a simple strategy aimed at isolating and characterizing a traditionally underrepresented protein class. Membrane protein profiles are created that discriminate between microsomal fractions of breast epithelial cells in different stages of neoplastic progression. Proteins are subsequently identified using matrix‐assisted laser desorption/ionization – mass spectrometry (MALDI‐MS) mass fingerprinting and MALDI‐quadrupole time of flight – tandem mass spectrometry (QTOF‐MS/MS) peptide sequencing. Furthermore, as this strategy preserves intact protein structure, further characterization can be performed on proteins producing mass fingerprint spectra and fragmentation spectra that did not result in database protein identifications. The coupling of nonporous RP‐HPLC with SDS‐PAGE provides a useful alternative to two‐dimensional PAGE (2‐D‐PAGE) for membrane protein analysis.
High‐throughput functional affinity purification of mannose binding proteins from Oryza sativa
Authors: Andon Nancy L. , Eckert Donna , Yates John R. , Haynes Paul A. 
Published: July 2003

AbstractWe have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using α‐D‐mannose as the ligand. Bound fractions were eluted and subjected to one‐dimensional electrophoresis, followed by high‐performance liquid chromatography‐tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue‐specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high‐throughput fashion.
Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results
Authors: Masselon Christophe , Paša‐Tolić Ljiljana , Lee Sang‐Won , Li Lingjun , Anderson Gordon A. , Harkewicz Richard , Smith Richard D. 
Published: July 2003

AbstractMultiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three “y” or “b” fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform‐ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.
The human serum proteome: Display of nearly 3700 chromatographically separated protein spots on two‐dimensional electrophoresis gels and identification of 325 distinct proteins
Authors: Pieper Rembert , Gatlin Christine L. , Makusky Anthony J. , Russo Paul S. , Schatz Courtney R. , Miller Stanton S. , Su Qin , McGrath Andrew M. , Estock Marla A. , Parmar Prashanth P. , Zhao Ming , Huang Shih‐Ting , Zhou Jeff , Wang Fang , Esquer‐Blasco Ricardo , Anderson N. Leigh , Taylor John , Steiner Sandra 
Published: July 2003

AbstractPlasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme‐linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two‐dimensional electrophoresis (2‐DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion‐exchange and size‐exclusion chromatography. Serum proteins from 74 fractions were displayed on 2‐DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post‐translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brillant Blue G‐250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin‐6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2‐DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.