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Laser Desorption/ionization-mass Spectrometry (laser + desorption_spectrometry)
Selected AbstractsGlycoproteomics of N -glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: Application to congenital disorders of glycosylationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2005Dijana Abstract A general strategy for the structural evaluation of N -glycosylation, a common post-translational protein modification, is presented. The methods for the release of N -linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N -glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N -glycans was compared. The most favourable results for native acidic N -glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N -glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented. [source] Integrated selective enrichment target , a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseasesELECTROPHORESIS, Issue 21-22 2004Simon Ekström Abstract The performance of a miniaturized sample processing platform for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), manufactured by silicon microfabrication, called integrated selective enrichment target (ISET) technology was evaluated in a biological context. The ISET serves as both sample treatment device and MALDI-MS target, and contains an array of 96 perforated nanovials, which each can be filled with 40 nL of reversed-phase beads. This methodology minimizes the number of sample transfers and the total surface area available for undesired adsorption of the analytes in order to provide high-sensitivity analysis. ISET technology was successfully applied for characterization of proteins coisolated by affinity chromatography of prostate-specific antigen (PSA) from human seminal fluid. The application of ISET sample preparation enabled multiple analyses to be performed on a limited sample volume, which resulted in the discovery that prolactin inducible protein (PIP) was coisolated from the samples. [source] Electronic gel protein transfer and identification using matrix-assisted laser desorption/ionization-mass spectrometryELECTROPHORESIS, Issue 9 2004Jonathan W. Cooper Abstract An electronic protein transfer technique is described for achieving the rapid and efficient recovery of sodium dodecyl sulfate (SDS)-protein complexes from polyacrylamide gels. This process involves the use of small-dimension capillaries in physical contact with a resolved protein band within the polyacrylamide gel, providing a large potential drop and high electric field strength at the capillary/gel interface. Several factors controlling the electronic protein transfer, including the applied electric field strength, the electrophoresis buffer concentration, and the capillary dimension, are studied to further enhance the use of field-amplification for sample stacking of extracted SDS-protein complexes. As a result of sample stacking, the extracted proteins from a 50 ng gel loading are present in a narrow (,80 nL) and highly concentrated (0.46 mg/mL or 3.3×10,5 M for cytochrome c) solution plug. Three model proteins with molecular mass ranging from 14 kDa (cytochrome c) to 116 kDa (,-galactosidase) are stained by Coomassie blue and electrophoretically extracted from gels with protein loadings as low as 50 ng. The capillary format of the electronic protein transfer technique allows direct deposition of extracted proteins onto a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target. Various matrices and solvent compositions are evaluated for the analysis of extracted and concentrated SDS-protein complexes using MALDI-MS. The electronic protein transfer technique, when operated under optimized conditions, is demonstrated for the effective (>70% recovery), speedy (less than 5 min), and sensitive MS identification of gel resolved proteins (as low as 50 ng). [source] Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometryELECTROPHORESIS, Issue 7-8 2004Jung-Kap Choi Abstract A fast and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon (ZC) and ethyl violet (EV) to form an ion-pair complex. The protocol, including fixing, staining and quick washing steps, can be completed in 1,1.5 h depending upon gel thickness. It has a sensitivity of 4,8 ng, comparable to that of colloidal Coomassie Brilliant Blue G (CBBG) staining with phosphoric acid in the staining solution. The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from MS. Considering the speed, sensitivity and compatibility with MS, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches. [source] Comparison of automated in-gel digest methods for femtomole level samplesELECTROPHORESIS, Issue 19-20 2003Erin J. Finehout Abstract A comparison of automated in-gel digestion methods for low picomolar to femtomolar levels of protein is presented. Gel spots with 4 pmol to 120 fmol of protein were stained with either Coomassie colloidal blue or SYPRO Ruby and digested using an automated platform. The sequence coverages and average peak intensities obtained from a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis are compared. Results show that methods using an acetonitrile extraction or digest times greater than the standard 4 h give no significant increase in peptide sequence coverage for automated digestion of low protein level samples. It is also shown that digests from SYPRO Ruby-stained gels show a greater improvement upon ZipTip cleanup than digests from Coomassie colloidal blue-stained gels. The digests from SYPRO Ruby-stained gels are also shown to give a higher average peptide intensity if a method with minimal gel plug washing is used. [source] Thiol-reactive dyes for fluorescence labeling of proteomic samplesELECTROPHORESIS, Issue 14 2003Kamala Tyagarajan Abstract Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA and BODIPY Fl C1 -IA were highly specific for cysteine residues and showed little or no nonspecific labeling even at very high dye:thiol ratios. These dyes also showed minimal effects on pI's of standard proteins. Some iodoacetamide dyes, (5-TMRIA and eosin-5-iodoacetamide) and some maleimide dyes (ThioGlo I and Rhodamine Red C2 maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2-carboxyethyl)phosphine (TCEP); interactions between TCEP and dye were also observed. Thiourea, an important component of sample solubilization cocktails, inhibited labeling of proteins with iodoacetamide dyes but not with maleimide dyes. Maleimide dyes may serve as an alternative for labeling proteins where it is essential to have thiourea in the solubilization buffer. Covalent derivatization by BODIPY TMR cadaverine IA, BODIPY Fl C1 -IA or Rhodamine Red C2 maleimide was also demonstrated to be compatible with in-gel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionization-mass spectrometry and allowed successful protein identification. [source] Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrixassisted laser desorption/ionization-mass spectrometry for identification of disease-related proteinsELECTROPHORESIS, Issue 24 2002Jina Kim Abstract Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3,10 gels, and also images for soluble fraction separated on pH 4,7 and pH 6,9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5,17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4,7 map and 95 spots in pH 6,9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed. [source] Multivariate analysis approach to the plasma protein profile of patients with advanced colorectal cancer,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2006Eugenio Ragazzi Abstract The aim of the present study was to identify the pattern of plasma protein species of interest as markers of colorectal cancer (CRC). Using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), the plasma protein profile was determined in nine stage IV CRC patients (study group) and nine clean-colon healthy subjects (control group). Multivariate analysis methods were employed to identify distinctive disease patterns at protein spectrum. In the study and control groups, cluster analysis (CA) on the complete MALDI-MS spectra plasma protein profile showed a distinction between CRC patients and healthy subjects, thus allowing the identification of the most discriminating ionic species. Principal component analysis (PCA) and linear discriminant analysis (LDA) yielded similar grouping results. LDA with leave-one-out cross validation achieved a correct classification rate of 89% in both the patients and the healthy subjects. Copyright © 2006 John Wiley & Sons, Ltd. [source] A strategy for high-resolution protein identification in surface-enhanced laser desorption/ionization mass spectrometry: Calgranulin A and chaperonin 10 as protein markers for endometrial carcinomaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2005Jingzhong Guo Abstract Surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) has conventionally been practiced on linear time of flight (TOF) which has low mass accuracy and resolution. Here we demonstrate in an examination of both malignant and nonmalignant endometrial tissue homogenates that high mass accuracy and resolution in the MS stage are crucial. Using a commercially available quadrupole/TOF (QqTOF), we were able to resolve two potential cancer markers, subsequently identified off-line as chaperonin 10 and calgranulin A, that differ by 8 Da in mass. Two off-line protein identification protocols were developed: the first was based on size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein extraction, trypsin digestion, and matrix-assisted laser desorption/ionization-tandem MS (MALDI-MS/MS); the second on SEC and shotgun nano-liquid chromatography (nanoLC)-MS/MS. Analyses on a cohort of 44 endometrial homogenates showed 22 out of 23 nonmalignant samples had nondetectable to very low abundance of chaperonin 10 and calgranulin A; 17 of the 21 malignant samples had detectable to abundant levels of both proteins. Immunohistochemical staining of a tissue microarray of 32 samples showed that approximately half of malignant endometrial tissues exhibited positive staining for calgranulin A in the malignant epithelium, while 9 out of 10 benign tissues exhibited negative epithelial staining. In addition, macrophages/granulocytes in malignant as well as nonmalignant tissues showed positive staining. No immunostaining occurred in stroma or myometrium. Calgranulin A, in combination with chaperonin 10 and other proteins, may eventually constitute a panel of markers to permit diagnosis and screening of endometrial cancer. [source] Platinum Nanoflowers on Scratched Silicon by Galvanic Displacement for an Effective SALDI Substrate,CHEMISTRY - A EUROPEAN JOURNAL, Issue 35 2010Dr. Hideya Kawasaki Abstract We report a new and facile method for synthesizing 3D platinum nanoflowers (Pt Nfs) on a scratched silicon substrate by electroless galvanic displacement and discuss the applications of the Pt Nfs in surface-assisted laser desorption/ionization-mass spectrometry (SALDI-MS). Surface scratching of n-type silicon is essential to induce Pt Nf growth on a silicon substrate (to obtain a Pt Nf silicon hybrid plate) by the galvanic displacement reaction. The Pt Nf silicon hybrid plate showed excellent SALDI activity in terms of the efficient generation of protonated molecular ions in the absence of a citrate buffer. We propose that the acidity of the SiOH moieties on silicon increases because of the electron-withdrawing nature of the Pt Nfs; hence, proton transfer from the SiOH groups to the analyte molecules is enhanced, and finally, thermal desorption of the analyte ions from the surface occurs. Signal enhancement was observed for protonated molecular ions produced from a titania nanotube array (TNA) substrate on which Pt nanoparticles had been photochemically deposited. Moreover, surface modification of the Pt Nf silicon hybrid plate by perfluorodecyltrichlorosilane (FDTS) (to obtain an FDTS-Pt Nf silicon hybrid plate) was found to facilitate soft SALDI of labile compounds. More interestingly, the FDTS-Pt Nf silicon hybrid plate acts 1),as a high-affinity substrate for phosphopeptides and 2),as a SALDI substrate. The feasibility of using the FDTS-Pt Nf silicon hybrid plate for SALDI-MS has been demonstrated by using a ,-casein digest and various analytes, including small molecules, peptides, phosphopeptides, phospholipids, carbohydrates, and synthetic polymers. The hybridization of Pt Nfs with a scratched silicon substrate has been found to be important for achieving excellent SALDI activity. [source] | ||||||||||