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Koichi Tanaka - One of the best experts on this subject based on the ideXlab platform.
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improvement of mass spectrometry analysis of glycoproteins by maldi ms using 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid
Analytical and Bioanalytical Chemistry, 2013Co-Authors: Makoto Watanabe, Kazuya Terasawa, Kaoru Kaneshiro, Hiromasa Uchimura, Rie Yamamoto, Yuko Fukuyama, Kazuharu Shimizu, Takaaki Sato, Koichi TanakaAbstract:Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-Hydroxycinnamic Acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-Hydroxycinnamic Acid or 2,5-dihydroxybenzoic Acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.
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an optimized matrix assisted laser desorption ionization sample preparation using a liquid matrix 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid for phosphopeptides
Rapid Communications in Mass Spectrometry, 2012Co-Authors: Yuko Fukuyama, Shinichi Iwamoto, Kohei Takeyama, Shinichirou Kawabata, Koichi TanakaAbstract:RATIONALE A liquid matrix, 3-aminoquinoline (3-AQ)/α-cyano-4-Hydroxycinnamic Acid (CHCA), introduced by Kolli et al. in 1996 for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported for peptides and proteins, oligonucleotides, oligosaccharides, and glycopeptides. However, it has not been validated for phosphopeptides. METHODS We optimized sample preparation using 3-AQ/CHCA for phosphopeptides. The sensitivity of six phosphopeptide species as isolated or in digests was systematically evaluated by using MALDI-quadropole ion trap (QIT)-time of flight (TOF) MS in positive and negative ion modes, and compared with the conventional methods using a solid matrix, 2,5-dihydroxybenzoic Acid (2,5-DHB). RESULTS The sensitivity of mono- and tetraphosphopeptides was improved 10- to 10 000-fold with the optimized preparation method using 3-AQ/CHCA compared with the conventional methods using 2,5-DHB. Improvement by 3-AQ/CHCA itself was 10-fold. Adding ammonium dihydrogen phosphate or an analyte solvent composition was also effectively improved the sensitivity. Phosphopeptides in isolated form or in digests were detected at femto- or subfemtomole levels. CONCLUSIONS Sensitivity of phosphopeptides was improved by the optimized sample preparation method using 3-AQ/CHCA compared with the conventional method using 2,5-DHB. The validation of 3-AQ/CHCA for phosphopeptides was systematically confirmed, expanding the potential of this matrix to phosphoproteomics. Copyright © 2012 John Wiley & Sons, Ltd.
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rapid quantitative profiling of n glycan by the glycan labeling method using 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid
Analytical Chemistry, 2012Co-Authors: Kaoru Kaneshiro, Makoto Watanabe, Kazuya Terasawa, Hiromasa Uchimura, Yuko Fukuyama, Kazuharu Shimizu, Takaaki Sato, Shinichi Iwamoto, Gozoh Tsujimoto, Koichi TanakaAbstract:Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-Hydroxycinnamic Acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R2 > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile da...
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on target separation of analyte with 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid liquid matrix for matrix assisted laser desorption ionization mass spectrometry
Rapid Communications in Mass Spectrometry, 2012Co-Authors: Sadanori Sekiya, Kenichi Taniguchi, Koichi TanakaAbstract:3-Aminoquinoline/α-cyano-4-Hydroxycinnamic Acid (3AQ/CHCA) is a liquid matrix (LM), which was reported by Kumar et al. in 1996 for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It is a viscous liquid and has some advantages of durability of ion generation by a self-healing surface and quantitative performance. In this study, we found a novel aspect of 3AQ/CHCA as a MALDI matrix, which converges hydrophilic material into the center of the droplet of analyte-3AQ/CHCA mixture on a MALDI sample target well during the process of evaporation of water derived from analyte solvent. This feature made it possible to separate not only the buffer components, but also the peptides and oligosaccharides from one another within 3AQ/CHCA. The MALDI imaging analyses of the analyte-3AQ/CHCA droplet indicated that the oligosaccharides and the peptides were distributed in the center and in the whole area around the center of 3AQ/CHCA, respectively. This 'on-target separation' effect was also applicable to glycoprotein digests such as ribonuclease B. These features of 3AQ/CHCA liquid matrix eliminate the requirement for pretreatment, and reduce sample handling losses thus resulting in the improvement of throughput and sensitivity. Copyright © 2012 John Wiley & Sons, Ltd.
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highly sensitive maldi analyses of glycans by a new aminoquinoline labeling method using 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid liquid matrix
Analytical Chemistry, 2011Co-Authors: Kaoru Kaneshiro, Sadanori Sekiya, Yuko Fukuyama, Shinichi Iwamoto, Koichi TanakaAbstract:In glycomics, mass spectrometry is an indispensable tool for high throughput analyses. Generally speaking, glycans contain many hydroxyl groups and are more difficult to ionize than peptides. Derivatization of glycans has been useful for increasing sensitivity. However, it takes time to purify and causes loss of sample. Here, we show a highly sensitive aminoquinoline (AQ)-labeling method of glycans on a matrix-assisted laser desorption/ionization (MALDI) target using a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-Hydroxycinnamic Acid (CHCA). It is a rapid procedure and reduces loss of sample material during the reaction process, especially in negative ion mode where 10 amol of monosialylated N-glycan were detected as AQ-labeled molecular ions. In addition, MS/MS of 10 amol of monosialylated N-glycan was achieved.
Mitsuo Takayama - One of the best experts on this subject based on the ideXlab platform.
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disappearance of interfering alkali metal adducted peaks from matrix assisted laser desorption ionization mass spectra of peptides with serine addition to alpha cyano 4 Hydroxycinnamic Acid matrix
Rapid Communications in Mass Spectrometry, 2007Co-Authors: Takashi Nishikaze, Mitsuo TakayamaAbstract:It has been described that ion yield in both positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of peptides is often inhibited by trace amounts of alkali metals and that the MALDI mass spectra are contaminated by the interfering peaks originating from traces of alkali metals, even when sample preparation is carefully performed. Addition of serine to the commonly used MALDI matrix alpha-cyano-4-Hydroxycinnamic Acid (CHCA) significantly improved and enhanced the signals of both protonated and deprotonated peptides, [M+H](+) and [M-H](-). The addition of serine to CHCA matrix eliminated the alkali-metal ion adducts, [M+Na](+) and [M+K](+), and the CHCA cluster ions from the mass spectra. Serine and serinephosphate as additives to CHCA enhanced and improved the formation of molecular-related ions of phosphopeptides in negative-ion MALDI mass spectra.
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disappearance of interfering alkali metal adducted peaks from matrix assisted laser desorption ionization mass spectra of peptides with serine addition to alpha cyano 4 Hydroxycinnamic Acid matrix
Rapid Communications in Mass Spectrometry, 2007Co-Authors: Takashi Nishikaze, Mitsuo TakayamaAbstract:It has been described that ion yield in both positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of peptides is often inhibited by trace amounts of alkali metals and that the MALDI mass spectra are contaminated by the interfering peaks originating from traces of alkali metals, even when sample preparation is carefully performed. Addition of serine to the commonly used MALDI matrix α-cyano-4-Hydroxycinnamic Acid (CHCA) significantly improved and enhanced the signals of both protonated and deprotonated peptides, [M+H]+ and [M−H]−. The addition of serine to CHCA matrix eliminated the alkali-metal ion adducts, [M+Na]+ and [M+K]+, and the CHCA cluster ions from the mass spectra. Serine and serinephosphate as additives to CHCA enhanced and improved the formation of molecular-related ions of phosphopeptides in negative-ion MALDI mass spectra. Copyright © 2007 John Wiley & Sons, Ltd.
Yuko Fukuyama - One of the best experts on this subject based on the ideXlab platform.
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improvement of mass spectrometry analysis of glycoproteins by maldi ms using 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid
Analytical and Bioanalytical Chemistry, 2013Co-Authors: Makoto Watanabe, Kazuya Terasawa, Kaoru Kaneshiro, Hiromasa Uchimura, Rie Yamamoto, Yuko Fukuyama, Kazuharu Shimizu, Takaaki Sato, Koichi TanakaAbstract:Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-Hydroxycinnamic Acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-Hydroxycinnamic Acid or 2,5-dihydroxybenzoic Acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.
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an optimized matrix assisted laser desorption ionization sample preparation using a liquid matrix 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid for phosphopeptides
Rapid Communications in Mass Spectrometry, 2012Co-Authors: Yuko Fukuyama, Shinichi Iwamoto, Kohei Takeyama, Shinichirou Kawabata, Koichi TanakaAbstract:RATIONALE A liquid matrix, 3-aminoquinoline (3-AQ)/α-cyano-4-Hydroxycinnamic Acid (CHCA), introduced by Kolli et al. in 1996 for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported for peptides and proteins, oligonucleotides, oligosaccharides, and glycopeptides. However, it has not been validated for phosphopeptides. METHODS We optimized sample preparation using 3-AQ/CHCA for phosphopeptides. The sensitivity of six phosphopeptide species as isolated or in digests was systematically evaluated by using MALDI-quadropole ion trap (QIT)-time of flight (TOF) MS in positive and negative ion modes, and compared with the conventional methods using a solid matrix, 2,5-dihydroxybenzoic Acid (2,5-DHB). RESULTS The sensitivity of mono- and tetraphosphopeptides was improved 10- to 10 000-fold with the optimized preparation method using 3-AQ/CHCA compared with the conventional methods using 2,5-DHB. Improvement by 3-AQ/CHCA itself was 10-fold. Adding ammonium dihydrogen phosphate or an analyte solvent composition was also effectively improved the sensitivity. Phosphopeptides in isolated form or in digests were detected at femto- or subfemtomole levels. CONCLUSIONS Sensitivity of phosphopeptides was improved by the optimized sample preparation method using 3-AQ/CHCA compared with the conventional method using 2,5-DHB. The validation of 3-AQ/CHCA for phosphopeptides was systematically confirmed, expanding the potential of this matrix to phosphoproteomics. Copyright © 2012 John Wiley & Sons, Ltd.
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rapid quantitative profiling of n glycan by the glycan labeling method using 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid
Analytical Chemistry, 2012Co-Authors: Kaoru Kaneshiro, Makoto Watanabe, Kazuya Terasawa, Hiromasa Uchimura, Yuko Fukuyama, Kazuharu Shimizu, Takaaki Sato, Shinichi Iwamoto, Gozoh Tsujimoto, Koichi TanakaAbstract:Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-Hydroxycinnamic Acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R2 > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile da...
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highly sensitive maldi analyses of glycans by a new aminoquinoline labeling method using 3 aminoquinoline α cyano 4 Hydroxycinnamic Acid liquid matrix
Analytical Chemistry, 2011Co-Authors: Kaoru Kaneshiro, Sadanori Sekiya, Yuko Fukuyama, Shinichi Iwamoto, Koichi TanakaAbstract:In glycomics, mass spectrometry is an indispensable tool for high throughput analyses. Generally speaking, glycans contain many hydroxyl groups and are more difficult to ionize than peptides. Derivatization of glycans has been useful for increasing sensitivity. However, it takes time to purify and causes loss of sample. Here, we show a highly sensitive aminoquinoline (AQ)-labeling method of glycans on a matrix-assisted laser desorption/ionization (MALDI) target using a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-Hydroxycinnamic Acid (CHCA). It is a rapid procedure and reduces loss of sample material during the reaction process, especially in negative ion mode where 10 amol of monosialylated N-glycan were detected as AQ-labeled molecular ions. In addition, MS/MS of 10 amol of monosialylated N-glycan was achieved.
Takashi Nishikaze - One of the best experts on this subject based on the ideXlab platform.
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disappearance of interfering alkali metal adducted peaks from matrix assisted laser desorption ionization mass spectra of peptides with serine addition to alpha cyano 4 Hydroxycinnamic Acid matrix
Rapid Communications in Mass Spectrometry, 2007Co-Authors: Takashi Nishikaze, Mitsuo TakayamaAbstract:It has been described that ion yield in both positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of peptides is often inhibited by trace amounts of alkali metals and that the MALDI mass spectra are contaminated by the interfering peaks originating from traces of alkali metals, even when sample preparation is carefully performed. Addition of serine to the commonly used MALDI matrix alpha-cyano-4-Hydroxycinnamic Acid (CHCA) significantly improved and enhanced the signals of both protonated and deprotonated peptides, [M+H](+) and [M-H](-). The addition of serine to CHCA matrix eliminated the alkali-metal ion adducts, [M+Na](+) and [M+K](+), and the CHCA cluster ions from the mass spectra. Serine and serinephosphate as additives to CHCA enhanced and improved the formation of molecular-related ions of phosphopeptides in negative-ion MALDI mass spectra.
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disappearance of interfering alkali metal adducted peaks from matrix assisted laser desorption ionization mass spectra of peptides with serine addition to alpha cyano 4 Hydroxycinnamic Acid matrix
Rapid Communications in Mass Spectrometry, 2007Co-Authors: Takashi Nishikaze, Mitsuo TakayamaAbstract:It has been described that ion yield in both positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of peptides is often inhibited by trace amounts of alkali metals and that the MALDI mass spectra are contaminated by the interfering peaks originating from traces of alkali metals, even when sample preparation is carefully performed. Addition of serine to the commonly used MALDI matrix α-cyano-4-Hydroxycinnamic Acid (CHCA) significantly improved and enhanced the signals of both protonated and deprotonated peptides, [M+H]+ and [M−H]−. The addition of serine to CHCA matrix eliminated the alkali-metal ion adducts, [M+Na]+ and [M+K]+, and the CHCA cluster ions from the mass spectra. Serine and serinephosphate as additives to CHCA enhanced and improved the formation of molecular-related ions of phosphopeptides in negative-ion MALDI mass spectra. Copyright © 2007 John Wiley & Sons, Ltd.
Michael Karas - One of the best experts on this subject based on the ideXlab platform.
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Graphite Supported Preparation (GSP) of α-Cyano-4-Hydroxycinnamic Acid (CHCA) for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) for Peptides and Proteins
Journal of The American Society for Mass Spectrometry, 2012Co-Authors: Jan Gorka, Ute Bahr, Michael KarasAbstract:Graphite as MALDI matrix or in combination with other substances has been reported in recent years. Here, we demonstrate that graphite can be used as target coating supporting the crystallization of the α-cyano-4-Hydroxycinnamic Acid matrix. A conventional dried-droplet preparation of matrix and analyte solution on a graphite-coated metal target leads to a thin, uniform layer of cubic crystals with about 1 μm edge length. Commercially available graphite powder of 1–2 μm particle size is gently wiped over the target using a cotton Q-tip, leading to an ultra-thin, not-visible film. This surface modification considerably improves analysis of peptides and proteins for MALDI MS using conventional dried-droplet preparation. Compared with untreated targets, the signal intensities of standard peptides are up to eight times higher when using the graphite supported crystallization. The relative standard deviation in peak area of angiotensin II for sample amounts between 1 and 50 fmol is reduced to about 15 % compared with 45 % for untreated sample holders. For a quantification of 1 fmol of the peptide using an internal standard the coefficient of variation is reduced to 3.5 % from 8 %. The new graphite supported preparation (GSP) protocol is very simple and does not require any technical nor manual skills. All standard solvents for peptides and proteins can be used.
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Comparison between the Matrices α-Cyano-4-Hydroxycinnamic Acid and 4-Chloro-α-cyanocinnamic Acid for Trypsin, Chymotrypsin, and Pepsin Digestions by MALDI-TOF Mass Spectrometry
Journal of proteome research, 2009Co-Authors: Thorsten W. Jaskolla, Dimitrios G. Papasotiriou, Michael KarasAbstract:The performance of the recently developed 4-chloro-α-cyanocinnamic Acid (Cl-CCA) matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) matrix was investigated in comparison to the most widely used matrix α-cyano-4-Hydroxycinnamic Acid (CHCA). For this purpose, in-solution digestions of standard proteins in the low femtomole range with the proteases trypsin, chymotrypsin, and pepsin were used as analytes. For all protein−protease combinations, Cl-CCA revealed to be highly superior in terms of number of identified peptides, obtained sequence coverages and peptide detection reproducibility. A deeper inspection of the detected peptide signals with regard to both physicochemical peptide properties (their isoelectric point) and mass spectrometric performance (signal-to-noise ratios and mass accuracies) showed that the progress achieved with Cl-CCA is due to the detection of numerous Acidic to neutral peptides. Moreover, the higher Cl-CCA sensitivity allowed for the detection of numerous addit...
