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Laser Desorption/ionization (laser + ionization)
Kinds of Laser Desorption/ionization Terms modified by Laser Desorption/ionization Selected AbstractsPROTEOLYTIC ACTIVITY OF LACTOBACILLUS SAKEI, LACTOBACILLUS FARCIMINIS AND LACTOBACILLUS PLANTARUM ON SARCOPLASMIC PROTEINS OF PORK LEANJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2004ANNA LISA BASSO The aim of this study was to assess the proteolytic activity of Lactobacillus sakei (DSM 6333), L. plantarum (B21), and to a lesser extent, L. farciminis (DSM 20184) on meat sarcoplasmic proteins. The protein composition was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis after incubation of meat extract inoculated with bacteria. All strains showed proteolytic activity: a band about 94 kDa disappeared in samples inoculated with L. farciminis and L. plantarum and strongly decreased in those inoculated with L. sakei. The intensity of the bands with a molecular weight between 94 and 38 kDa decreased in all samples. Capillary electrophoresis analysis ascertained the disappearance of the fractions corresponding to 8.64 and 8.66 min retention time in all samples. The bands corresponding to 94 kDa and 38 kDa were, respectively, identified as glycogen phosphorylase muscle isoform and glyceraldehyde 3-phosphate dehydrogenase, by in situ digestion of protein gel bands and peptide map analysis using Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS). [source] Comparison of two glutaraldehyde immobilization techniques for solid-phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometryELECTROPHORESIS, Issue 9 2004Isabelle Migneault Abstract Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively. [source] Actinide-Transition Metal Heteronuclear Ions and Their Oxides: {IrUO}+ as an Analogue to UranylEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 17 2006Marta Santos Abstract Recent theoretical calculations have shown that Ir should behave as a chemical analogue to N, with the result that IrUO+, like known NUO+, is predicted to be a stable species isoelectronic with UO22+, the uranyl dication. The target heterometallic analogue to uranyl has now been prepared by direct laser desorption/ionization of a U/Ir alloy, and by oxidation of UIr+ with N2O and C2H4O. Properties of UIr+, UPt+, and UAu+ bimetallic ions have been studied. They demonstrate direct actinide,transition metal bonding, and support the concept of "autogenic isolobality". (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Alterations in Barrett's-related adenocarcinomas: A proteomic approachINTERNATIONAL JOURNAL OF CANCER, Issue 6 2008DunFa Peng Abstract In this study, we applied high-resolution, two-dimensional, gel electrophoresis and matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry analysis (MALDI TOF MS) to identify novel proteins that are involved in Barrett's tumorigenesis. We analyzed 12 primary tissue samples that included 8 Barrett's-related adenocarcinomas (BA) and 4 normal mucosae samples. Twenty-three spots were consistently altered (,2-fold) in at least half of the tumors when compared with all normal samples and thus subjected to further analysis. The MALDI TOF MS analysis demonstrated biologically interesting upregulated proteins such as ErbB3, Dr5 and Cyclin D1 as well as several members of the zinc finger proteins (Znf146, Znf212 and Znf363). Examples of downregulated proteins included Lgi1 and Klf6. We selected four proteins (ErbB3, Dr5, Znf146 and Lgi1) that are novel for BAs for validation using quantitative real-time reverse-transcription PCR on 39 BA tissue samples when compared with normal samples. We demonstrated mRNA upregulation of ERBB3 (51.3%), DR5 (41%) and ZNF146 (30.7%) and downregulation of LGI1 (100%) in BA. We have further validated the protein overexpression of ErbB3, Dr5 and Znf146, using immunohistochemical (IHC) analysis on a tissue microarray that contained 75 BAs and normal gastric and esophageal mucosae samples. BA tissue samples demonstrated overexpression of ErbB3 (42%), Dr5 (90%) and Znf146 (30%) when compared with normal tissues. In conclusion, we have identified and validated several novel proteins that are involved in Barrett's carcinogenesis. © 2007 Wiley-Liss, Inc. [source] The Detection of Multiply Charged Dyes Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for the Forensic Examination of Pen Ink Dyes Directly from PaperJOURNAL OF FORENSIC SCIENCES, Issue 5 2007Jamie D. Dunn M.S. Abstract:, Laser desorption mass spectrometry (LDMS) is emerging as a technique for questioned document examination. Its use is limited to detecting ink dyes that are neutral or singly charged. Several inks contain dyes that are multiply charged and LDMS cannot be employed for their identification. We have successfully detected >20 polyionic dyes that can be used in the manufacture of inks using matrix-assisted laser desorption/ionization (MALDI) MS, directly from paper, with the matrix, 2-(4-hydroxyphenylazo)benzoic acid (HABA), and the additive, diammonium hydrogen citrate (DAHC). For example, Acid Violet 49, a charged dye containing one positively-charged site and two negatively charged sulfonate groups, cannot be detected by LDMS, but forms intact, singly charged ions in the MALDI MS experiment. The method described is also useful for identifying multiply charged dye mixtures that are used in modern pen inks. [source] Parental relationships among three grape varieties studied by MALDI of grape seed protein profiles,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2010Antonella Bertazzo Abstract Two Raboso cultivars, i.e. Raboso Veronese and Raboso Piave, are two black Vitis vinifera varieties. A genetic study suggested that Raboso Veronese is the progeny of a spontaneous cross between Raboso Piave and Marzemina Bianca cultivars. Parental relationships are usually investigated by genetic studies, which are effective to establish genetic links among different cultivars. Considering that proteome is the genome expression, in this article we evaluated the power of seed protein profiles obtained by matrix-assisted laser desorption/ionization (MALDI)/MS for parentage investigation. The three cultivars lead to very similar spectra with differences in the relative intensity of the most abundant species and the presence of very weak specific ions. In order to evaluate the analytical significance of these aspects, the variability due to instrumental factors and due to different harvesting areas and years of the same cultivars have been considered and measured by the calculation of discrepancy factor values. On one hand, the results obtained can be considered a valid confirmation of the genomic findings, whereas on the other hand, the results provide evidence for the ability of MALDI/MS to individuate minor differences in protein profiles of complex protein mixtures. Copyright © 2010 John Wiley & Sons, Ltd. [source] Molecular dynamics simulations of MALDI: laser fluence and pulse width dependence of plume characteristics and consequences for matrix and analyte ionizationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2010Richard Knochenmuss Abstract Molecular dynamics simulations of matrix-assisted laser desorption/ionization were carried out to investigate laser pulse width and fluence effects on primary and secondary ionization process. At the same fluence, short (35 or 350 ps) pulses lead to much higher initial pressures and ion concentrations than longer ones (3 ns), but these differences do not persist because the system relaxes toward local thermal equilibrium on a nanosecond timescale. Higher fluences accentuate the initial disparities, but downstream differences are not substantial. Axial velocities of ions and neutrals are found to span a wide range, and be fluence dependent. Total ion yield is only weakly dependent on pulse width, and consistent with experimental estimates. Secondary reactions of matrix cations with analyte neutrals are efficient even though analyte ions are ablated in clusters of matrix. Copyright © 2010 John Wiley & Sons, Ltd. [source] Laser desorption postionization for imaging MS of biological materialJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2010Artem Akhmetov Abstract Vacuum ultraviolet single photon ionization (VUV SPI) is a soft ionization technique that has the potential to address many of the limitations of matrix-assisted laser desorption/ionization (MALDI) for imaging MS. Laser desorption postionization (LDPI) uses VUV SPI for postionization and is experimentally analogous to a MALDI instrument with the addition of a pulsed VUV light source. This review discusses progress in LDPI-MS over the last decade, with an emphasis on imaging MS of bacterial biofilms, analytes whose high salt environment make them particularly resistant to imaging by MALDI-MS. This review first considers fundamental aspects of VUV SPI including ionization mechanisms, cross sections, quantum yields of ionization, dissociation and potential mass limits. The most common sources of pulsed VUV radiation are then described along with a newly constructed LDPI-MS instrument with imaging capabilities. Next, the detection and imaging of small molecules within intact biofilms is demonstrated by LDPI-MS using 7.87 eV (157.6 nm) VUV photons from a molecular fluorine excimer laser, followed by the use of aromatic tags for detection of selected species within the biofilm. The final section considers the future prospects for imaging intact biological samples by LDPI-MS. Copyright © 2010 John Wiley & Sons, Ltd. [source] Matrix-assisted laser desorption/ionization tissue profiling of secretoneurin in the nucleus accumbens shell from cocaine-sensitized ratsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2010Joachim D. Uys Abstract Proteins in the nucleus accumbens mediate many cocaine-induced behaviors. In an effort to measure changes in nucleus accumbens protein expression as potential biomarkers for addiction, coronal tissue sections were obtained from rats that developed behavioral sensitization after daily administration of cocaine, or from daily saline-treated controls. The tissue sections were subjected to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) profiling and tissue imaging. For profiling experiments, brain sections were manually spotted with matrix over the nucleus accumbens, a brain region known to regulate cocaine sensitization. Summed mass spectra (10 000 laser shots, grid) were acquired and spectra were aligned to reference peaks. Using bioinformatics tools, eight spectral features were found to be altered by cocaine treatment. Based on additional sequencing experiments with MALDI tandem MS and database searches of measured masses, secretoneurin (m/z 3653) was identified as having an increased expression. In addition, the distribution of m/z 3653 in the nucleus accumbens was determined by MALDI tissue imaging, and the increased expression of its precursor protein, secretogranin II, was verified by immunoblotting. Copyright © 2009 John Wiley & Sons, Ltd. [source] A convenient purification and preconcentration of peptides with ,-cyano-4-hydroxycinnamic acid matrix crystals in a pipette tip for matrix-assisted laser desorption/ionization mass spectrometry,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2010Helena, ehulková Abstract Peptide samples derived from enzymatic in-gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed-phase pipette tip purification, on-target washing, adding co-matrices, etc. As a suitable matrix for MALDI MS of peptides, ,-cyano-4-hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on-target washing of peptide samples can significantly improve MALDI MS signals. Although the common on-target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on-target washing principle carried out in a narrow-end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA-tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed. Copyright © 2009 John Wiley & Sons, Ltd. [source] From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics.JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2009Part II 198 Abstract This is the continuation of a personal retrospective on the developments that since 1965 have given shape to Mass Spectrometry (MS) and taken it from a position of simply playing a role in Protein Chemistry to becoming an indispensable tool in Proteomics, all within a 40-year span. Part I covered the period from 1965 to 1984. This second part reviews the Mass Spectrometry timeline of events from 1985 to 2000, stopping at various time points where MS made significant contributions to protein chemistry or where the development of new instrumentation for MS represented a major advance for peptide and protein work. Major highlights in the field and their significance for peptide and protein characterization such as the advent and practical consequences of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are covered, including work done with triple quads, the development of time-of-flight (TOF) instruments and new ion traps and going on to the more recent work on the full characterization of the Proteome with ion traps, TOF instruments and new ionization and tagging techniques for protein sequencing. Copyright © 2009 John Wiley & Sons, Ltd. [source] Microfluidic chips for mass spectrometry-based proteomicsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2009Jeonghoon Lee Abstract Microfluidic devices coupled to mass spectrometers have emerged as excellent tools for solving the complex analytical challenges associated with the field of proteomics. Current proteome identification procedures are accomplished through a series of steps that require many hours of labor-intensive work. Microfluidics can play an important role in proteomic sample preparation steps prior to mass spectral identification such as sample cleanup, digestion, and separations due to its ability to handle small sample quantities with the potential for high-throughput parallel analysis. To utilize microfluidic devices for proteomic analysis, an efficient interface between the microchip and the mass spectrometer is required. This tutorial provides an overview of the technologies and applications of microfluidic chips coupled to mass spectrometry for proteome analysis. Various approaches for combining microfluidic devices with electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are summarized and applications of chip-based separations and digestion technologies to proteomic analysis are presented. Copyright © 2009 John Wiley & Sons, Ltd. [source] Modern MALDI time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2009Marvin L. Vestal Abstract This paper focuses on development of time-of-flight (TOF) mass spectrometry in response to the invention of matrix-assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI-TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high-performance MALDI-TOF and TOF-TOF with off-line high-capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC-MS and MS-MS. Copyright © 2009 John Wiley & Sons, Ltd. [source] Evaluation of a new matrix-free laser desorption/ionization method through statistic studies: comparison of the DIAMS (desorption/ionization on self-assembled monolayer surface) method with the MALDI and TGFA-LDI techniquesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2008Matthieu Bounichou Abstract This work demonstrates that the desorption/ionization on self-assembled monolayer surface (DIAMS) mass spectrometry, a recent matrix-free laser desorption/ionization (LDI) method based on an organic target plate, is as statistically repeatable and reproducible as matrix assisted laser desorption ionization (MALDI) and thin gold film-assisted laser desorption/ionization (TGFA-LDI) mass spectrometries. On lipophilic DIAMS of target plates with a mixture of glycerides, repeatability/reproducibility has been estimated at 15 and 30% and the relative detection limit has been evaluated at 0.3 and 3 pmol, with and without NaI respectively. Salicylic acid and its d6 -isomer analysis confirm the applicability of the DIAMS method in the detection of compounds of low molecular weight. Copyright © 2008 John Wiley & Sons, Ltd. [source] A sialylation study of mouse brain gangliosides by MALDI a-TOF and o-TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2008Mostafa Zarei Abstract Matrix-assisted laser desorption/ionization (MALDI) process of sialoglycoconjugates is generally accompanied by different levels of cleavage of sialic acid residues and/or by dehydration, and decarboxylation reactions. Quantitative densitometry of the mouse brain ganglioside (MBG) components separated by high-performance thin layer chromatography (HPTLC) and evidenced by orcinol staining was a basis to verify the ganglioside composition pattern with respect to the relative abundances of individual components in the mixture. A systematic mass spectrometry (MS) sialylation analysis has been carried out to evaluate the feasibility of an axial time-of-flight (a-TOF) MS, equipped with a vacuum MALDI source and an orthogonal-TOF (o-TOF) instrument with an ion source operated at about 1 mbar of N2. Besides, the esterification by one methyl group of the carboxyl group in sialic acid to increase the stability of the ganglioside species for MALDI MS analysis has been tested and the yield of intact ganglioside species and of the neutral loss of water and carbon dioxide estimated. For the sialylation analysis of native ganglioside mixtures the MALDI o-TOF analysis with 6-azo-2-thiothymine/diammonium citrate (ATT/DAC) as a matrix appears as an optimal approach for ganglioside profiling. Copyright © 2008 John Wiley & Sons, Ltd. [source] Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2007M. Reid Groseclose Abstract A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain. Copyright © 2007 John Wiley & Sons, Ltd. [source] Direct laser desorption/ionization time-of-flight mass spectrometry of conjugated polymersJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2007Zhun Ma Abstract Two conjugated polymers (CPs), poly(9,9-dioctylfluorene) (PF) and poly(3-octylthiophene) (PT) were analyzed by direct laser desorption/ionization time-of-flight mass spectrometry (LDI-ToF MS). Because of their strong absorption near the wavelength of the laser (337 nm), easy and transient energy transfer properties and sufficient thermal stability, CPs can be desorbed and ionized directly without a matrix. For comparison, these two polymers were also analyzed using matrix-assisted laser desorption/ionization (MALDI)-ToF MS in the positive reflectron mode. The results revealed that they are very similar in terms of quality and resolution. All results demonstrate that LDI-ToF MS is an alternative method for the mass characterization of some conjugated systems, thereby simplifying the process of sample preparation and result analysis. Copyright © 2006 John Wiley & Sons, Ltd. [source] Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2006Tomoya Kinumi Abstract Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, ,-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid. Copyright © 2005 John Wiley & Sons, Ltd. [source] Measurement of serum salicylate levels by solid-phase extraction and desorption/ionization on silicon mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2005Shoji Okuno Abstract The applicability of the matrix-free laser desorption/ionization on silicon mass spectrometry (DIOS-MS) to measuring serum drug levels was examined by analyzing serum salicylic acid. The optimized and simple solid-phase extraction (SPE) allowed good recovery, 88.9 ± 5.8%, for 1.4 mM (200 mg/L) of salicylic acid in serum. The negative ion MS allowed measurements of deprotonated molecules without interference from other signals. Using a deuterium-labeled internal standard, good linearity was obtained in the 0.14 to 4.2 mM (20,600 mg/L) range, which was sufficient for monitoring the therapeutic anti-inflammatory dose. SPE followed by DIOS-MS is anticipated to be a method of measuring drug levels in blood and may allow high throughput analysis. Copyright © 2005 John Wiley & Sons, Ltd. [source] Glycan side chains on naturally presented MHC class II ligandsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2005Jörn Dengjel Abstract The molecular characterization of unknown naturally presented major histocompatibility complex (MHC) class II glycopeptides carrying complex glycans has so far not been achieved, reflecting the different fragmentation characteristics of sugars and peptides in mass spectrometric analysis. Human leukocyte antigen (HLA)-DR-bound peptides were isolated by affinity purification, separated via high performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. We were able to identify two naturally processed MHC class II ligands, CD53122,136 and CD53121,136, carrying complex N -linked glycan side chains by a combination of in-source and collision-induced fragmentation on a quadrupole time-of-flight tandem mass spectrometer. Copyright © 2005 John Wiley & Sons, Ltd. [source] Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2004Martin Zehl Abstract A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H]2+ ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme. Copyright © 2004 John Wiley & Sons, Ltd. [source] Study of peptide,sugar non-covalent complexes by infrared atmospheric pressure matrix-assisted laser desorption/ionizationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2004Christopher E. Von Seggern Abstract Infrared atmospheric pressure matrix-assisted laser desorption/ionization quadrupole ion trap mass spectrometry was applied to the study of siglec binding to oligosaccharide ligands. Peptides were designed to mimic the binding sites of three members of the siglec family: sialoadhesin, MAG and CD22. These peptides were tested for their ability to complex with their carbohydrate ligands 3, -sialyllactose (3,SL) and 6, -sialyllactose (6,SL). All peptides demonstrated the ability to bind to the carbohydrates, with the peptide representing sialoadhesin maintaining its binding specificity for 3,SL in preference to 6,SL. This technique can be used to study other protein,sugar interactions and can be expanded to create high-throughput screening techniques. Copyright © 2004 John Wiley & Sons, Ltd. [source] Matrix-assisted laser desorption/ionization collision-induced dissociation of linear single oligomers of nylon-6JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2001Renata Murgasova Abstract Matrix-assisted laser desorption/ionization, collision induced-dissociation (MALDI-CID) has been used to obtain structural information for linear single oligomers of nylon-6. The effects of matrix and cationization agent in MALDI-CID analysis have been investigated. Fragmentation mechanisms are proposed for the series of ions that are observed in the MALDI-CID spectra of the hexamer, octamer and dodecamer. Fragmentation processes observed in the MALDI-CID spectra include cleavage of the end groups followed by dissociation of the m/z 113 unit. Cleavage of the oligamide chain occurs at the amide linkage, as well as at adjacent bonds. For the four matrices and three cationization agents investigated, 2,5-dihydroxybenzoic acid and sodium chloride showed the best performance for MALDI-CID analysis of the dodecamer. In addition, yields of the fragment ions in MALDI-CID spectra were found to be dependent on the chain length distribution. Copyright © 2001 John Wiley & Sons, Ltd. [source] Characterization by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry of the major photoproducts of temoporfin (m -THPC) and bacteriochlorin (m -THPBC)JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2001Marc Angotti Abstract The photobleaching of 5,10,15,20-tetrakis(m -hydroxyphenyl)chlorin (temoporfin, m -THPC) and 5,10,15,20-tetrakis(m -hydroxyphenyl)bacteriochlorin (bacteriochlorin, m -THPBC) was studied in ethanol,water (1 : 99, v/v) and in physiological medium (phosphate-buffered saline, PBS) with or without fetal calf serum (FCS). m -THPC solution was irradiated with the laser radiation of 650 nm, whereas m -THPBC solution underwent two consecutive irradiations at 532 and 650 nm. The photoproducts were characterized by UV,visible absorption spectrophotometry and by matrix-assisted laser desorption/ionization (MALDI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Independent of the solvent used, the phototransformation of either photosensitizer yielded the formation of 5,10,15,20-tetrakis (m -hydroxyphenyl)porphyrin (m -THPP) through a major dehydrogenation process. Copyright © 2001 John Wiley & Sons, Ltd. [source] Investigation of tyrosine nitration in proteins by mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2001Ann-Sofi Petersson Abstract In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites. Copyright © 2001 John Wiley & Sons, Ltd. [source] Electrospray and matrix-assisted laser desorption/ionization mass spectral characterization of linear single nylon-6 oligomersJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2001Lu Shan Abstract Synthetic nylon-6 single molecular mass oligomers were studied by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. These oligomers, considered as model compounds for the study of nylon-6 polymers, gave good mass spectrometric results using both MALDI and ESI. In spite of the gentle nature of both techniques, the MALDI and ESI spectra showed evidence of end-group cleavage from the oligomer chains. MALDI-MS was found to give similar fragmentation patterns for all of the oligomer samples. An increase in doubly charged ion signals with increasing oligomer mass was observed in the ESI mass spectra, as was end-group fragmentation. Signals from oligomer clusters were observed in ESI-MS for the dimer, tetramer and hexamer, most likely due to non-covalent bonding among the low-mass oligomer molecules. Copyright © 2001 John Wiley & Sons, Ltd. [source] Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis with mass spectrometryJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Cyrus P. Billimoria Abstract Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI,TOF (matrix-assisted laser desorption/ionization,time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified. [source] 71 Proteomics of haematococcus pluvialis: new opportunities for study of genomics of a non-sequenced speciesJOURNAL OF PHYCOLOGY, Issue 2003Q. Hu The green alga, Haematococcus pluvialis, has become a model organism for commercial production of the high-value carotenoid astaxanthin. H. Pluvialis has also drawn significant scientific attention because fundamental biological questions relating to the massive cellular accumulation of astaxanthin have to be addressed in order to improve the yield and quality of the algal biomass. However, research has been impeded by the lack of molecular background information on this non-sequenced species. A combination of classical biochemistry with a state-of-the-art proteomic approach was used to address these questions. This was possible by taking advantage of information already available for homologous genes/gene-products in organisms whose genomes have been sequenced. The approach involved isolation of subsets of the proteome from subcellular compartments/organelles of an organism by one- or two-dimensional electrophoresis (1-DE or 2-DE) and their identification by N-terminal sequencing and peptide mass fingerprinting (PMF), involving matrix-assisted laser desorption/ionization and time-of-flight (MALDI-TOF) mass spectrometry coupled with bioinformatics. Based upon the information obtained from the combined methods, expression and physiological functions of specific genes/encoded proteins may be deduced. Examples include profiling of cell wall proteins, biogenesis and protein composition of lipid bodies, and expression patterns of soluble proteins under stress conditions. Advantages and limitations of the method for non-sequenced organisms and for cross-species protein identification will also be discussed. [source] Synthesis and characterization of macrocyclic vinyl aromatic polymersJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 7 2006Thieo E. Hogen-Esch Abstract The synthesis and characterization by size exclusion chromatography, liquid chromatography, NMR, matrix-assisted laser desorption/ionization, thermal analysis, and other techniques of well-defined and narrow molecular weight distribution macrocyclic polystyrene (PS), poly(2-vinylpyridine), poly(,-methylstyrene), poly (2-vinyl-naphthalene) (P2VN), and poly(9,9-dimethyl-2-vinylfluorene) (PDMVF) containing a single 1,4-benzylidene, methylidene, or 9,10-anthracenylidene unit are reviewed. The absorption and emission spectroscopy of PS, P2VN, and PDMVF is also discussed. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2139,2155, 2006 [source] Oncoproteomics of hepatocellular carcinoma: from cancer markers' discovery to functional pathwaysLIVER INTERNATIONAL, Issue 8 2007Stella Sun Abstract Hepatocellular carcinoma (HCC) is a heterogeneous cancer with no promising treatment and remains one of the most prevailing and lethal malignancies in the world. Researchers in many biological areas now routinely identify and characterize protein markers by a mass spectrometry-based proteomic approach, a method that has been commonly used to discover diagnostic biomarkers for cancer detection. The proteomic research platforms span from the classical two-dimensional polyacrylamide gel electrophoresis (2-DE) to the latest Protein Chip or array technology, which are often integrated with the MALDI (matrix-assisted laser-desorption ionization), SELDI (surface-enhanced laser desorption/ionization) or tandem mass spectrometry (MS/MS). New advances on quantitative proteomic analysis (e.g. SILAC, ICAT, and ITRAQ) and multidimensional protein identification technology (MudPIT) have greatly enhanced the capability of proteomic methods to study the expressions, modifications and functions of protein markers. The present article reviews the latest proteomic development and discovery of biomarkers in HCC that may provide insights into the underlying mechanisms of hepatocarcinogenesis and the readiness of biomarkers for clinical uses. [source] | ||||||||||||||||||||||||||||||||||