scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

Implantable neural interfaces

2018 ◽  
Author(s):  
Stefano Vassanelli
Keyword(s):  
Brain Function ◽  
Large Scale ◽  
Ionic Channels ◽  
Patch Clamp Technique ◽  
Advantages And Disadvantages ◽  
Optogenetic Stimulation ◽  
Physical Interfaces ◽  
The Brain

Establishing direct communication with the brain through physical interfaces is a fundamental strategy to investigate brain function. Starting with the patch-clamp technique in the seventies, neuroscience has moved from detailed characterization of ionic channels to the analysis of single neurons and, more recently, microcircuits in brain neuronal networks. Development of new biohybrid probes with electrodes for recording and stimulating neurons in the living animal is a natural consequence of this trend. The recent introduction of optogenetic stimulation and advanced high-resolution large-scale electrical recording approaches demonstrates this need. Brain implants for real-time neurophysiology are also opening new avenues for neuroprosthetics to restore brain function after injury or in neurological disorders. This chapter provides an overview on existing and emergent neurophysiology technologies with particular focus on those intended to interface neuronal microcircuits in vivo. Chemical, electrical, and optogenetic-based interfaces are presented, with an analysis of advantages and disadvantages of the different technical approaches.

scholarly journals Shedding new light on opsin evolution

2011 ◽  
Vol 279 (1726) ◽  
pp. 3-14 ◽  
Author(s):  
Megan L. Porter ◽  
Joseph R. Blasic ◽  
Michael J. Bok ◽  
Evan G. Cameron ◽  
Thomas Pringle ◽  
...  
Keyword(s):  
Evolutionary History ◽  
Large Scale ◽  
Current Model ◽  
Expression Patterns ◽  
Model Organisms ◽  
Biological Functions ◽  
Site Specific ◽  
History Of ◽  
Functional Features

Opsin proteins are essential molecules in mediating the ability of animals to detect and use light for diverse biological functions. Therefore, understanding the evolutionary history of opsins is key to understanding the evolution of light detection and photoreception in animals. As genomic data have appeared and rapidly expanded in quantity, it has become possible to analyse opsins that functionally and histologically are less well characterized, and thus to examine opsin evolution strictly from a genetic perspective. We have incorporated these new data into a large-scale, genome-based analysis of opsin evolution. We use an extensive phylogeny of currently known opsin sequence diversity as a foundation for examining the evolutionary distributions of key functional features within the opsin clade. This new analysis illustrates the lability of opsin protein-expression patterns, site-specific functionality (i.e. counterion position) and G-protein binding interactions. Further, it demonstrates the limitations of current model organisms, and highlights the need for further characterization of many of the opsin sequence groups with unknown function.

scholarly journals Advantages and disadvantages of conditional systems for characterization of essential genes in Toxoplasma gondii

Parasitology ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 1390-1398 ◽  
Author(s):  
ELENA JIMÉNEZ-RUIZ ◽  
ELEANOR H. WONG ◽  
GURMAN S. PALL ◽  
MARKUS MEISSNER
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
Gene Function ◽  
Function Analysis ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Advantages And Disadvantages ◽  
Gene Function Analysis ◽  
Tetracycline Inducible System

SUMMARYThe dissection of apicomplexan biology has been highly influenced by the genetic tools available for manipulation of parasite DNA. Here, we describe different techniques available for the generation of conditional mutants. Comparison of the advantages and disadvantages of the three most commonly used regulation systems: the tetracycline inducible system, the regulation of protein stability and site-specific recombination are discussed. Using some previously described examples we explore some of the pitfalls involved in gene-function analysis using these systems that can lead to wrong or over-interpretation of phenotypes. We will also mention different options to standardize the application of these techniques for the characterization of gene function in high-throughput.

scholarly journals Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

2019 ◽  
Author(s):  
Nicholas M. Riley ◽  
Alexander S. Hebert ◽  
Michael S. Westphall ◽  
Joshua J. Coon
Keyword(s):  
Large Scale ◽  
Electron Transfer Dissociation ◽  
Protein Glycosylation ◽  
Analytical Challenge ◽  
Post Translational Modification ◽  
Structural Domains ◽  
Site Specific ◽  
Future Studies ◽  
Site Heterogeneity ◽  
Degree Of Heterogeneity

ABSTRACTProtein glycosylation is a highly important, yet a poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity and analytical challenge. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has ultimately limited our abilities to both address the degree of heterogeneity across the glycoproteome and to understand how it contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling with methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Moreover, we have used this large-scale glycoproteomic data to develop several new visualizations that will prove useful for analyzing intact glycopeptides in future studies. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein.

scholarly journals Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

2021 ◽  
Vol 11 (18) ◽  
pp. 8314
Author(s):  
Jozafina Milicaj ◽  
Colleen D. Castro ◽  
Nadiya Jaunbocus ◽  
Erika A. Taylor
Keyword(s):  
Bacterial Infections ◽  
Visual Cues ◽  
Large Scale ◽  
Lipid A ◽  
Biosynthetic Enzymes ◽  
Bacterial Cells ◽  
Site Specific ◽  
E Coli ◽  
Key Steps

The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

Some applications of electron beam microanalysis techniques in VLSI process evaluation

1983 ◽  
Vol 41 ◽  
pp. 160-161
Author(s):  
Simon Thomas
Keyword(s):  
Integrated Circuits ◽  
Process Evaluation ◽  
Large Scale ◽  
Technology Development ◽  
Analytical Techniques ◽  
Successful Implementation ◽  
Analysis Of Failures ◽  
Characterization Of Materials ◽  
Circuits Vlsi

Trends in the technology development of very large scale integrated circuits (VLSI) have been in the direction of higher density of components with smaller dimensions. The scaling down of device dimensions has been not only laterally but also in depth. Such efforts in miniaturization bring with them new developments in materials and processing. Successful implementation of these efforts is, to a large extent, dependent on the proper understanding of the material properties, process technologies and reliability issues, through adequate analytical studies. The analytical instrumentation technology has, fortunately, kept pace with the basic requirements of devices with lateral dimensions in the micron/ submicron range and depths of the order of nonometers. Often, newer analytical techniques have emerged or the more conventional techniques have been adapted to meet the more stringent requirements. As such, a variety of analytical techniques are available today to aid an analyst in the efforts of VLSI process evaluation. Generally such analytical efforts are divided into the characterization of materials, evaluation of processing steps and the analysis of failures.

Sign in / Sign up

Export Citation Format

Share Document