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David M Lubman - One of the best experts on this subject based on the ideXlab platform.
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separation of tryptic digests using a modified buffer in pressurized capillary electrochromatography with an ion trap storage reflectron time of flight mass spectrometer
Analytical Chemistry, 1998Co-Authors: Peiqing Huang, And Jingtaowu, David M LubmanAbstract:A rapid and efficient separation method using pressurized capillary electrochromatography (pCEC) has been developed to separate protein digests. The effects of mobile-phase ion conductivity, pH, and column size on separation speed and column efficiency were studied and optimized. The pCEC method was demonstrated to provide enhanced speed with more efficient and selective separation than HPLC by performing separations of a bovine β-lactoglobulin A digest. A human hemoglobin digest was separated by pCEC under optimized conditions, and more than 20 peaks were separated in less than 20 min. Using an ion trap storage/reflectron time-of-flight mass spectrometer, coeluting components were clearly identified, and the resolving power of the pCEC method was further enhanced.
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protein digest analysis by pressurized capillary electrochromatography using an ion trap storage reflectron time of flight mass detector
Analytical Chemistry, 1997Co-Authors: Peiqing Huang, David M LubmanAbstract:Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogr...
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use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on line detection via an ion trap storage reflectron time of flight mass spectrometer
Analytical Chemistry, 1997Co-Authors: Michael X Li, Jing Tao Wu, David M LubmanAbstract:A polybrene capillary coating in capillary electrophoresis (CE) has been used for rapid analysis of hemoglobin variant digests. The use of the polybrene capillary coating has allowed sufficient separation to resolve the large number of digest products formed upon tryptic digestion of the whole protein, so that prior separation of the hemoglobin α and β chains is not required. The resolution of the digest peaks obtained by CE is sufficient so that even single amino acid substitutions can easily be detected using UV absorption detection. The digest is further analyzed by capillary electrophoresis separation with on-line detection using electrospray ionization interfaced to the ion trap storage/reflectron time of flight device (CE/ESI-IT/reTOF), where a comparison of the total ion electropherograms and mass spectra of the mutant and normal hemoglobins can detect the presence of a mutation site. The CE separation and mass analysis can be accomplished in typically 10−15 min. The unique capability of the CE/ESI...
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use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on line detection via an ion trap storage reflectron time of flight mass spectrometer
Analytical Chemistry, 1997Co-Authors: Lin Liu, David M LubmanAbstract:A polybrene capillary coating in capillary electrophoresis (CE) has been used for rapid analysis of hemoglobin variant digests. The use of the polybrene capillary coating has allowed sufficient separation to resolve the large number of digest products formed upon tryptic digestion of the whole protein, so that prior separation of the hemoglobin α and β chains is not required. The resolution of the digest peaks obtained by CE is sufficient so that even single amino acid substitutions can easily be detected using UV absorption detection. The digest is further analyzed by capillary electrophoresis separation with on-line detection using electrospray ionization interfaced to the ion trap storage/reflectron time of flight device (CE/ESI-IT/reTOF), where a comparison of the total ion electropherograms and mass spectra of the mutant and normal hemoglobins can detect the presence of a mutation site. The CE separation and mass analysis can be accomplished in typically 10−15 min. The unique capability of the CE/ESI...
Norman J. Dovichi - One of the best experts on this subject based on the ideXlab platform.
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capillary zone electrophoresis multiple reaction monitoring from 100 pg of raw 264 7 cell lysate digest
Analyst, 2013Co-Authors: Yihan Li, Matthew M Champion, Roza Wojcik, Norman J. DovichiAbstract:Capillary zone electrophoresis-multiple/single reaction monitoring (CZE-MRM/SRM), which employed an electrokinetically driven sheath-flow electrospray interface, was used for the rapid and highly sensitive detection of protein analytes in complex tryptic digests. MRM channels were developed against a commercial exponential mixture of bovine proteins. Five proteins spanning four orders of magnitude concentration range were confidently detected from only 2.5 ng of the digest mixture; the mass detection limits (S/N = 3) of two detected proteins, alpha-casein and glutamate dehydrogenase were about 600 zmol and 30 amol, respectively. This technique was then applied to a RAW 264.7 cell lysate digest. Three proteins were confidently and reproducibly detected from 100 pg of this digest. The sample amount corresponds to the approximate protein content from a single cell, which suggests that CZE-MRM may be a useful analytical tool in chemical cytometry. In addition to providing highly sensitive detection of proteins in complex mixtures, this system is highly rapid; migration time of the protein digests was less than 10 min.
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single shot proteomics using capillary zone electrophoresis electrospray ionization tandem mass spectrometry with production of more than 1 250 escherichia coli peptide identifications in a 50 min separation
Analytical Chemistry, 2013Co-Authors: Norman J. DovichiAbstract:Capillary zone electrophoresis (CZE)–electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was optimized and applied for analysis of 1–100 ng Escherichia coli protein digests in a single run (single-shot analysis). The system employed an electrokinetically pumped nanospray interface, a coated capillary, and stacking conditions for sample injection. More than 1 250 peptides were identified by optimized single-shot CZE–ESI-MS/MS with 100 ng digest loaded and 50 min analysis time. When 10 ng and 1 ng digests were loaded, about 1 000 and 600 peptides were identified in a single-shot analysis, respectively. Compared with single-shot ultraperformance liquid chromatography (UPLC)–ESI-MS/MS, CZE–ESI-MS/MS produced fewer peptide IDs (1 377 ± 128 vs 1 875 ± 32) for large sample loading amounts (100 ng) with the same mass spectrometer time (50 min). However, when the loaded digest was mass limited (1 ng), CZE–ESI-MS/MS generated many more peptide identifications than UPLC–ESI-MS/MS (627 ± 38 vs 342 ± 113). In...
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on line amino acid based capillary isoelectric focusing esi ms ms for protein digests analysis
Analytica Chimica Acta, 2012Co-Authors: Guijie Zhu, Liangliang Sun, Ping Yang, Norman J. DovichiAbstract:Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300-1800. Good focusing was achieved for insulin receptor, which produced ~10 s peak width. For 0.1 mg mL(-1) bovine serum albumin (BSA) digests, 24±1 peptides (sequence coverage 47±4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.
Kenneth G Standing - One of the best experts on this subject based on the ideXlab platform.
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maldi quadrupole time of flight mass spectrometry a powerful tool for proteomic research
Analytical Chemistry, 2000Co-Authors: Andrej Shevchenko, Alexander Loboda, Anna Shevchenko, Kenneth G StandingAbstract:A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins c...
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maldi quadrupole time of flight mass spectrometry a powerful tool for proteomic research
Analytical Chemistry, 2000Co-Authors: Andrej Shevchenko, Alexander Loboda, Anna Shevchenko, Werner Ens, Kenneth G StandingAbstract:A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins can be identified in all types of sequence databases, including an EST database. Thus, MALDI QqTOF mass spectrometry promises to have remarkable potential for advancing proteomic research.
Andrej Shevchenko - One of the best experts on this subject based on the ideXlab platform.
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maldi quadrupole time of flight mass spectrometry a powerful tool for proteomic research
Analytical Chemistry, 2000Co-Authors: Andrej Shevchenko, Alexander Loboda, Anna Shevchenko, Kenneth G StandingAbstract:A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins c...
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maldi quadrupole time of flight mass spectrometry a powerful tool for proteomic research
Analytical Chemistry, 2000Co-Authors: Andrej Shevchenko, Alexander Loboda, Anna Shevchenko, Werner Ens, Kenneth G StandingAbstract:A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins can be identified in all types of sequence databases, including an EST database. Thus, MALDI QqTOF mass spectrometry promises to have remarkable potential for advancing proteomic research.
Peiqing Huang - One of the best experts on this subject based on the ideXlab platform.
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separation of tryptic digests using a modified buffer in pressurized capillary electrochromatography with an ion trap storage reflectron time of flight mass spectrometer
Analytical Chemistry, 1998Co-Authors: Peiqing Huang, And Jingtaowu, David M LubmanAbstract:A rapid and efficient separation method using pressurized capillary electrochromatography (pCEC) has been developed to separate protein digests. The effects of mobile-phase ion conductivity, pH, and column size on separation speed and column efficiency were studied and optimized. The pCEC method was demonstrated to provide enhanced speed with more efficient and selective separation than HPLC by performing separations of a bovine β-lactoglobulin A digest. A human hemoglobin digest was separated by pCEC under optimized conditions, and more than 20 peaks were separated in less than 20 min. Using an ion trap storage/reflectron time-of-flight mass spectrometer, coeluting components were clearly identified, and the resolving power of the pCEC method was further enhanced.
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protein digest analysis by pressurized capillary electrochromatography using an ion trap storage reflectron time of flight mass detector
Analytical Chemistry, 1997Co-Authors: Peiqing Huang, David M LubmanAbstract:Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogr...