The Experts below are selected from a list of 384 Experts worldwide ranked by ideXlab platform
Gozzo Theresa - One of the best experts on this subject based on the ideXlab platform.
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Towards a Greater Understanding of Post-Translational Modification of Transcription Factor, ArsR, in Helicobacter pylori via Bottom-Up Proteomics: Methods Development for Electrospray and Nanospray Approaches
W&M ScholarWorks, 2017Co-Authors: Gozzo TheresaAbstract:Studies presented here work towards the analysis of post-translational modification of the essential ArsR transcription factor of Helicobacter pylori. A human health hazard, H. pylori contributes to the development of gastric cancer and peptic ulcers in its hosts. It is of interest to study the mechanisms that allow the bacterium to survive long term in the harsh stomach environment. The ArsRS signaling pathway mediates the repression of sabA, the gene coding for the adhesin protein, SabA, in acidic conditions. The repression is dependent on histidine kinase, ArsS, but independent of phosphorylation of ArsR at the canonical aspartic acid 52 (D52). To begin comparing the repression mechanism in wild-type and mutant (D52E or D52N) H. pylori under neutral and acidic conditions, preliminary bottom-up proteomic investigations were conducted via LC-MS/MS. Data-dependent mass spectrometric analyses of proteolytic (Asp-N and trypsin) digests of purified ArsR protein were performed with an ESI-LTQ linear ion trap. Ultimately, SEQUEST identified the possible phosphorylation sites, D47, D52, and D59, with high confidence from the CID product ion spectra of the purified ArsR peptides. A shotgun proteomic analysis of H. pylori (26695) was also conducted, but it was determined that the combined low-abundance of ArsR and low signal imparted by ESI preclude PTM analysis of similar shotgun samples. In addition, Nanospray and electrospray methods were developed for these and future analyses. Drastic improvements in ionization efficiency and signal were observed with the Nanospray ionization source in comparison to electrospray. Reliable Nano-Flow HPLC and Nanospray ionization will be essential for future proteomic studies and the continuation of this work
Karl Mechtler - One of the best experts on this subject based on the ideXlab platform.
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improved sensitivity in low input proteomics using micro pillar array based chromatography
bioRxiv, 2019Co-Authors: Johannes Stadlmann, Otto Hudecz, Gabriela Krssakova, Geert Van Raemdonck, Jeff Op De Beeck, Gert Desmet, Josef M Penninger, Paul Jacobs, Karl MechtlerAbstract:ABSTRACT Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, Nano-LC-ESI-MS/MS is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for Nano-Flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the µPAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used Nano-Flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, increase sensitivity in the analysis of low-input proteomics samples, and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional Nano-Flow HPLC columns.
Josef M Penninger - One of the best experts on this subject based on the ideXlab platform.
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improved sensitivity in low input proteomics using micro pillar array based chromatography
bioRxiv, 2019Co-Authors: Johannes Stadlmann, Otto Hudecz, Gabriela Krssakova, Geert Van Raemdonck, Jeff Op De Beeck, Gert Desmet, Josef M Penninger, Paul Jacobs, Karl MechtlerAbstract:ABSTRACT Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, Nano-LC-ESI-MS/MS is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for Nano-Flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the µPAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used Nano-Flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, increase sensitivity in the analysis of low-input proteomics samples, and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional Nano-Flow HPLC columns.
Johannes Stadlmann - One of the best experts on this subject based on the ideXlab platform.
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improved sensitivity in low input proteomics using micro pillar array based chromatography
bioRxiv, 2019Co-Authors: Johannes Stadlmann, Otto Hudecz, Gabriela Krssakova, Geert Van Raemdonck, Jeff Op De Beeck, Gert Desmet, Josef M Penninger, Paul Jacobs, Karl MechtlerAbstract:ABSTRACT Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, Nano-LC-ESI-MS/MS is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for Nano-Flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the µPAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used Nano-Flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, increase sensitivity in the analysis of low-input proteomics samples, and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional Nano-Flow HPLC columns.
Matthias Mann - One of the best experts on this subject based on the ideXlab platform.
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a new parallel high pressure packing system enables rapid multiplexed production of capillary columns
bioRxiv, 2021Co-Authors: Johannes B Mullerreif, Fynn M Hansen, Lisa Schweizer, Peter V Treit, Philipp E Geyer, Matthias MannAbstract:Abstract Reversed-phase high performance liquid chromatography (HPLC) is the most commonly applied peptide separation technique in mass spectrometry (MS)-based proteomics. Particle-packed capillary columns are predominantly used in Nano-Flow HPLC systems. Despite being the broadly applied standard for many years capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column production with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as driving fluid, our system replaces the traditional setup of helium pressured packing bombs. By using 10x multiplexing, we have reduced the production time to just under 2 minutes for several 50 cm columns with 1.9 µm particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters (ID) and length packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station. One sentence summary A newly constructed parallel high-pressure packing system enables the rapid multiplexed production of capillary columns.
