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Stephen A. Martin - One of the best experts on this subject based on the ideXlab platform.
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a new Delayed Extraction maldi tof ms ms for characterization of protein digests
2000Co-Authors: Marvin L. Vestal, Peter Juhasz, Wade M. Hines, Stephen A. MartinAbstract:Two-dimensional gel electrophoresis is commonly used to separate and visualize proteins present in complex mixtures such as cell lysates [1]. Digestion of selected spots by one or more endopeptidases followed by generation of peptide mass maps using MALDI-TOF MS is now widely accepted as the first step toward identifying and characterizing the proteins [2]. Following the development of Delayed Extraction techniques for MALDI [3–5], and improved sample preparation and clean-up methodology [6], this strategy is often successful at identifying proteins represented in a database [7] even when the proteins are present at low levels. In favorable cases protein identification is successful at the sub-femtomole level. When this approach fails, it is generally necessary to generate sequence data using either Edman degradation or MS-MS techniques. The MS-MS technique known as post-source decay (PSD) with MALDI-TOF [8] sometimes provides sufficient sequence information, but often the sensitivity and mass accuracy are inadequate and interpretation is difficult. Recent developments in electrospray ionization, such as nanospray [9] have dramatically improved the sensitivity of triple quadrupole and ion trap MS-MS, but these techniques are rather slow and tedious, and rapid, automated interpretation of data to provide reliable sequences is not yet routine. New instruments employing TOF analyzers in place of the third quadrupole in triple quadrupole MS-MS systems have very recently been described which provide improved resolution and mass accuracy in fragment ion measurements [10, 11].
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On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of Delayed Extraction time-of-flight mass spectra
Journal of the American Society for Mass Spectrometry, 1997Co-Authors: Peter Juhasz, Marvin L. Vestal, Stephen A. MartinAbstract:A novel method was developed to measure the initial velocity of ions generated by matrix-assisted laser desorption ionization (MALDI). It is shown both experimentally and theoretically that with a Delayed Extraction (DE) technique, the flight time of an ion changes linearly with Extraction delay. The initial velocity of the ion, a consequence of the desorption process, can be determined from the slope of this linear curve. Systematic study of the initial velocity was undertaken regarding its dependence on the matrix substance, molecular weight of the analyte, ion polarity, and wavelength of irradiation. It was found that the most important factor was the matrix material. Sinapinic acid and α-cyano-4-hydroxycinnamic acid matrices ejected slower peptide and protein ions than 2,5-dihydroxybenzoic acid or 3-hydroxypicolinic acid: ∼ 300 versus ∼ 550 m/s. Matrix ions themselves exhibited a similar order of initial velocities, but these were 15–40% higher than those of insulin ions. The molecular weight of protein samples (between 5 and 25 ku) was found to have little effect on the initial velocity, but for peptides below 5 ku a gradual transition was noted toward the velocity of the matrix ions. Also decreasing velocity with increasing molecular mass was observed for DNA samples in the 4–14-ku range. In the negative ion mode slightly lower velocities were observed than in the positive ion mode. No difference was found between 337- and 266-nm irradiation. Values of the initial velocities were used to correct systematic errors in the internal calibration observed in mass spectra with Delayed Extraction. These velocity corrections decrease mass errors substantially in the linear mode, in particular for multicomponent mixtures.
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Accurate mass measurements using MALDI-TOF with Delayed Extraction.
Journal of protein chemistry, 1997Co-Authors: Edward J Takach, Peter Juhasz, Marvin L. Vestal, Wade M. Hines, Dale H. Patterson, Arnold M. Falick, Stephen A. MartinAbstract:Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With Delayed Extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100–500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.
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sequencing oligonucleotides by exonuclease digestion and Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Analytical Biochemistry, 1996Co-Authors: Igor P Smirnov, Peter Juhasz, Stephen A. Martin, Mark T Roskey, Edward J Takach, Lawrence A HaffAbstract:A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with Delayed ion Extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with Delayed Extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with Delayed Extraction MALDI compared with standard MALDI-MS.
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dna sequencing by Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Mark T Roskey, Peter Juhasz, Stephen A. Martin, Igor P Smirnov, Edward J Takach, Lawrence A HaffAbstract:Abstract Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a Delayed ion Extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.
Lawrence A Haff - One of the best experts on this subject based on the ideXlab platform.
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single nucleotide polymorphism identification assays using a thermostable dna polymerase and Delayed Extraction maldi tof mass spectrometry
Genome Research, 1997Co-Authors: Lawrence A Haff, Igor P SmirnovAbstract:We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to Delayed-Extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.
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sequencing oligonucleotides by exonuclease digestion and Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Analytical Biochemistry, 1996Co-Authors: Igor P Smirnov, Peter Juhasz, Stephen A. Martin, Mark T Roskey, Edward J Takach, Lawrence A HaffAbstract:A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with Delayed ion Extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with Delayed Extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with Delayed Extraction MALDI compared with standard MALDI-MS.
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dna sequencing by Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Mark T Roskey, Peter Juhasz, Stephen A. Martin, Igor P Smirnov, Edward J Takach, Lawrence A HaffAbstract:Abstract Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a Delayed ion Extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.
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applications of Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry to oligonucleotide analysis
Analytical Chemistry, 1996Co-Authors: Peter Juhasz, Marvin L. Vestal, Lawrence A Haff, Igor P Smirnov, Mark T Roskey, Stephen A. MartinAbstract:A Delayed ion Extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation t...
Igor P Smirnov - One of the best experts on this subject based on the ideXlab platform.
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single nucleotide polymorphism identification assays using a thermostable dna polymerase and Delayed Extraction maldi tof mass spectrometry
Genome Research, 1997Co-Authors: Lawrence A Haff, Igor P SmirnovAbstract:We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to Delayed-Extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.
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sequencing oligonucleotides by exonuclease digestion and Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Analytical Biochemistry, 1996Co-Authors: Igor P Smirnov, Peter Juhasz, Stephen A. Martin, Mark T Roskey, Edward J Takach, Lawrence A HaffAbstract:A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with Delayed ion Extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with Delayed Extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with Delayed Extraction MALDI compared with standard MALDI-MS.
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dna sequencing by Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Mark T Roskey, Peter Juhasz, Stephen A. Martin, Igor P Smirnov, Edward J Takach, Lawrence A HaffAbstract:Abstract Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a Delayed ion Extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.
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applications of Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry to oligonucleotide analysis
Analytical Chemistry, 1996Co-Authors: Peter Juhasz, Marvin L. Vestal, Lawrence A Haff, Igor P Smirnov, Mark T Roskey, Stephen A. MartinAbstract:A Delayed ion Extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation t...
Peter Juhasz - One of the best experts on this subject based on the ideXlab platform.
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a new Delayed Extraction maldi tof ms ms for characterization of protein digests
2000Co-Authors: Marvin L. Vestal, Peter Juhasz, Wade M. Hines, Stephen A. MartinAbstract:Two-dimensional gel electrophoresis is commonly used to separate and visualize proteins present in complex mixtures such as cell lysates [1]. Digestion of selected spots by one or more endopeptidases followed by generation of peptide mass maps using MALDI-TOF MS is now widely accepted as the first step toward identifying and characterizing the proteins [2]. Following the development of Delayed Extraction techniques for MALDI [3–5], and improved sample preparation and clean-up methodology [6], this strategy is often successful at identifying proteins represented in a database [7] even when the proteins are present at low levels. In favorable cases protein identification is successful at the sub-femtomole level. When this approach fails, it is generally necessary to generate sequence data using either Edman degradation or MS-MS techniques. The MS-MS technique known as post-source decay (PSD) with MALDI-TOF [8] sometimes provides sufficient sequence information, but often the sensitivity and mass accuracy are inadequate and interpretation is difficult. Recent developments in electrospray ionization, such as nanospray [9] have dramatically improved the sensitivity of triple quadrupole and ion trap MS-MS, but these techniques are rather slow and tedious, and rapid, automated interpretation of data to provide reliable sequences is not yet routine. New instruments employing TOF analyzers in place of the third quadrupole in triple quadrupole MS-MS systems have very recently been described which provide improved resolution and mass accuracy in fragment ion measurements [10, 11].
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On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of Delayed Extraction time-of-flight mass spectra
Journal of the American Society for Mass Spectrometry, 1997Co-Authors: Peter Juhasz, Marvin L. Vestal, Stephen A. MartinAbstract:A novel method was developed to measure the initial velocity of ions generated by matrix-assisted laser desorption ionization (MALDI). It is shown both experimentally and theoretically that with a Delayed Extraction (DE) technique, the flight time of an ion changes linearly with Extraction delay. The initial velocity of the ion, a consequence of the desorption process, can be determined from the slope of this linear curve. Systematic study of the initial velocity was undertaken regarding its dependence on the matrix substance, molecular weight of the analyte, ion polarity, and wavelength of irradiation. It was found that the most important factor was the matrix material. Sinapinic acid and α-cyano-4-hydroxycinnamic acid matrices ejected slower peptide and protein ions than 2,5-dihydroxybenzoic acid or 3-hydroxypicolinic acid: ∼ 300 versus ∼ 550 m/s. Matrix ions themselves exhibited a similar order of initial velocities, but these were 15–40% higher than those of insulin ions. The molecular weight of protein samples (between 5 and 25 ku) was found to have little effect on the initial velocity, but for peptides below 5 ku a gradual transition was noted toward the velocity of the matrix ions. Also decreasing velocity with increasing molecular mass was observed for DNA samples in the 4–14-ku range. In the negative ion mode slightly lower velocities were observed than in the positive ion mode. No difference was found between 337- and 266-nm irradiation. Values of the initial velocities were used to correct systematic errors in the internal calibration observed in mass spectra with Delayed Extraction. These velocity corrections decrease mass errors substantially in the linear mode, in particular for multicomponent mixtures.
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Accurate mass measurements using MALDI-TOF with Delayed Extraction.
Journal of protein chemistry, 1997Co-Authors: Edward J Takach, Peter Juhasz, Marvin L. Vestal, Wade M. Hines, Dale H. Patterson, Arnold M. Falick, Stephen A. MartinAbstract:Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With Delayed Extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100–500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.
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sequencing oligonucleotides by exonuclease digestion and Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Analytical Biochemistry, 1996Co-Authors: Igor P Smirnov, Peter Juhasz, Stephen A. Martin, Mark T Roskey, Edward J Takach, Lawrence A HaffAbstract:A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with Delayed ion Extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with Delayed Extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with Delayed Extraction MALDI compared with standard MALDI-MS.
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dna sequencing by Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Mark T Roskey, Peter Juhasz, Stephen A. Martin, Igor P Smirnov, Edward J Takach, Lawrence A HaffAbstract:Abstract Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a Delayed ion Extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.
Tamotsu Taketomi - One of the best experts on this subject based on the ideXlab platform.
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evaluation of sphingolipids in vitreous bodies from a patient with gaucher disease using Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Journal of Chromatography B, 2004Co-Authors: Takehisa Fujiwaki, Seiji Yamaguchi, Masaru Tasaka, Masaki Takayanagi, Mariko Isobe, Tamotsu TaketomiAbstract:Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide in tissues. Using Delayed Extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS), we analyzed sphingolipids in vitreous bodies from a patient with Gaucher disease who suffered from vitreous opacities. Crude lipids were extracted from the freeze-dried vitreous bodies with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE MALDI-TOF-MS. The results were as follows: (a) the m/z values of the ions found in the mass spectra for both the control and the Gaucher disease patient corresponded to different sphingomyelin species. (b) The mass spectrum of the Gaucher disease patient showed additional ions with m/z values corresponding to different ceramide monohexoside (CMH) species. It was indicated that the accumulation of CMH in vitreous bodies from Gaucher disease patients could be easily detected with the DE MALDI-TOF-MS method.
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application of Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry for analysis of sphingolipids in pericardial fluid peritoneal fluid and serum from gaucher disease patients
Journal of Chromatography B, 2002Co-Authors: Takehisa Fujiwaki, Seiji Yamaguchi, Masaru Tasaka, Nobuo Sakura, Tamotsu TaketomiAbstract:Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide. Using Delayed Extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE-MALDI-TOF-MS), we analyzed sphingolipids in pericardial fluid, peritoneal fluid, and serum from two patients with Gaucher disease. Crude lipids were extracted from 1 ml each of pericardial fluid, peritoneal fluid, and serum with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE-MALDI-TOF-MS. The results were as follows: (a) in all the specimens, peaks of ceramide monohexoside and sphingomyelin were detected in both the controls and Gaucher disease patients; (b) in pericardial fluid, peritoneal fluid, and serum, the ceramide monohexoside/sphingomyelin ratio was increased in the Gaucher disease patients compared with in the controls. It was indicated that the accumulation of ceramide monohexoside in such samples from Gaucher disease patients can be easily detected with this DE-MALDI-TOF-MS method.
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avascular necrosis of the femoral head in a patient with fabry s disease identification of ceramide trihexoside in the bone by Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometry
Arthritis & Rheumatism, 2002Co-Authors: Hiroshi Horiuchi, Tamotsu Taketomi, Naoto Saito, Seneki Kobayashi, Hiroyoshi Ota, Kunio TakaokaAbstract:Fabry's disease is a lipid storage disease caused by an X-linked hereditary deficiency of alpha-galactosidase. The enzymatic defect causes progressive deposition of ceramide trihexoside (CTH) in various tissues, leading to renal failure, premature myocardial infarction, and stroke, with a high rate of mortality in younger patients. Among the complications associated with Fabry's disease, a few cases involving avascular necrosis (AVN) of the femoral head have been reported. However, direct evidence of deposition of CTH in bone marrow in the femoral head has not been demonstrated. This report describes a 58-year-old man who underwent total hip arthroplasty for femoral head AVN associated with Fabry's disease. The accumulation of CTH was examined by chemical analysis of the sphingolipid extracted from the femoral head, using Delayed-Extraction matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. This is the first report confirming the presence of CTH in the sphingolipid fraction from normal and necrotic bone of a patient with Fabry's disease.
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Application of Delayed Extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in cultured skin fibroblasts from sphingolipidosis patients.
Brain & Development, 2002Co-Authors: Takehisa Fujiwaki, Kazuko Sukegawa, Seiji Yamaguchi, Tamotsu TaketomiAbstract:Abstract Sphingolipidoses are caused by defects of enzymes involved in the hydrolysis of sphingolipids. Using Delayed Extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS), we analyzed sphingolipids in cultured skin fibroblasts from patients with sphingolipidoses, including: (a) Farber disease (FD, acid ceramidase deficiency); (b) Gaucher disease (GD); (c) Niemann-Pick disease type C (NPDC); and (d) GM1-gangliosidosis (GM1G). Crude lipids were extracted from about 50 mg wet weight of cultured skin fibroblasts. After mild alkaline treatment, a sphingolipid fraction was prepared from the crude lipids and analyzed by DE MALDI-TOF-MS. The results were as follows: (a) in fibroblasts from the FD patient, the ceramide/sphingomyelin and ceramide/monohexosylceramide ratios were both significantly high; (b) in the GD patient, the glucosylceramide/sphingomyelin ratio was increased; on the other hand; (c) in the NPDC patient, the monohexosylceramide/sphingomyelin ratio was within normal range; and (d) in the GM1G patient, no specific data were obtained. Sphingolipids in cultured fibroblasts can be evaluated by DE MALDI-TOF-MS, whereas GM1-ganglioside or its asialo derivatives are not detectable. With this DE MALDI-TOF-MS method, ceramide or monohexosylceramide accumulating in cultured fibroblasts from cases of sphingolipidoses, such as FD and GD, respectively, can be easily detected.
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preparation of various lysogangliosides including lyso fucosyl gm1 and Delayed Extraction matrix assisted laser desorption ionization time of flight mass spectrometric analysis
Journal of Biochemistry, 1997Co-Authors: Tamotsu Taketomi, Atsushi Hara, Kei-ichi Uemura, Hisashi Kurahashi, Eiko SugiyamaAbstract:Our rapid method of microwave-mediated saponification for preparing lysoglycosphingolipids from their parent glycosphingolipids was also able to prepare lysogangliosides or modified lysogangliosides, which were identified by Delayed Extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF MS) analysis. When GM3, GM2, and GM1 isolated from adult human brain gangliosides were subjected to the saponification, GM3 was found to give rise to only lyso-GM3 containing de-N-acetylneuraminic acid (de-N-acetyl lyso-GM3), whereas the GM2 produced both lyso-GM2 and the de-N-acetyl compound, and GM1 also gave both lyso-GM1 and the de-N-acetyl compound. In the saponification of GM1 and GDla, isolated from rat brain gangliosides, GM1 similarly produced both lyso-GM1 and the de-N-acetyl compound, but GDla was found to give rise to both dehydrated de-N-monoacetyl and dehydrated de-N-diacetyl lyso-GDla. However, the saponification of the GM1 fraction isolated from porcine brain gangliosides gave rise not only to both lyso-GM1 and the de-N-acetyl compound, but also unexpectedly to both lyso-fucosyl GM1 and its de-N-acetyl compound. The untreated GM1 fraction was examined by TLC and DE MALDI-TOF mass spectrometry, and proved to contain fucosyl-GM1. The DE MALDI-TOF MS analysis of the prepared lyso-gangliosides showed that their long chain bases consisted of d18:1 and d20:1 sphingosines in various ratios reflecting those of the different mammalian brain gangliosides.