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Daniel Kolarich - One of the best experts on this subject based on the ideXlab platform.
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glycan size and attachment site location affect electron transfer dissociation etd fragmentation and automated Glycopeptide identification
Glycoconjugate Journal, 2019Co-Authors: Kathirvel Alagesan, Hannes Hinneburg, Peter H Seeberger, Daniel Varon Silva, Daniel KolarichAbstract:We established a small synthetic N-Glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of electron transfer dissociation (ETD) fragmentation and subsequent automated identification. The Glycopeptides within this library differed in glycosylation site position and glycan size ranging from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans. Factors such as glycan size, glycosylation site position within a Glycopeptide and individual precursor m/z all significantly impacted the number and quality of assignable Glycopeptide backbone fragments. Generally, high charge/low m/z precursors (>3+) and Glycopeptides carrying neutral, smaller N-glycans gave better product ion spectra, while hardly any product ions were detectable for sialylated, triply charged N-Glycopeptides. These factors impacted correct Glycopeptide identification by proteomics software tools such as SEQUEST or Amanda. A better understanding how Glycopeptide physico-chemical properties influence fragmentation will help optimizing fragmentation conditions and generate better data, which will facilitate software assisted Glycopeptide data analyses.
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glycan size and attachment site location affect electron transfer dissociation etd fragmentation and automated Glycopeptide identification
bioRxiv, 2019Co-Authors: Kathirvel Alagesan, Hannes Hinneburg, Peter H Seeberger, Daniel Varon Silva, Daniel KolarichAbstract:We used a small synthetic Glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of ETD MS/MS fragmentation and subsequent automated identification. Understanding how the physico-chemical properties of Glycopeptides influence Glycopeptide fragmentation allows for optimizing fragmentation conditions and software-assisted data analyses, which rely on informative fragmentation spectra for subsequent data processing to identify Glycopeptides. Often, mis-assignment of Glycopeptides occurs due to uncertainties such as failure to produce sufficient peptide backbone fragment ions. Our synthetic Glycopeptide library contained Glycopeptides differing in glycosylation site position within the peptide as well as glycan size (from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans). Different software solutions such as SEQUEST and Amanda were compared for ETD Glycopeptide identification. We found that all, glycan size, glycosylation site position within a Glycopeptide and individual precursor m/z significantly impacted the number and quality of assignable Glycopeptide backbone fragments, and thus the likelihood to be correctly identified in software assisted data analyses.
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it is all about the solvent on the importance of the mobile phase for zic hilic Glycopeptide enrichment
Analytical and Bioanalytical Chemistry, 2017Co-Authors: Kathirvel Alagesan, Sana Khan Khilji, Daniel KolarichAbstract:Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on Glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed “Drop-HILIC”, which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) Glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic Glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC Glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct Glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic Glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic Glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic Glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC Glycopeptide enrichment and need to be carefully considered when employed for Glycopeptide enrichment.
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the art of destruction optimizing collision energies in quadrupole time of flight q tof instruments for Glycopeptide based glycoproteomics
Journal of the American Society for Mass Spectrometry, 2016Co-Authors: Hannes Hinneburg, Manfred Wuhrer, Peter H Seeberger, Daniel Varon Silva, Kathrin Stavenhagen, Ulrike Schweigerhufnagel, Stuart Pengelley, Wolfgang Jabs, Daniel KolarichAbstract:In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-Glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including Glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-Glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of Glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for Glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted Glycopeptide assignment of both N- and O-Glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-Glycopeptides and four out of five O-Glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based Glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.
Kathirvel Alagesan - One of the best experts on this subject based on the ideXlab platform.
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glycan size and attachment site location affect electron transfer dissociation etd fragmentation and automated Glycopeptide identification
Glycoconjugate Journal, 2019Co-Authors: Kathirvel Alagesan, Hannes Hinneburg, Peter H Seeberger, Daniel Varon Silva, Daniel KolarichAbstract:We established a small synthetic N-Glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of electron transfer dissociation (ETD) fragmentation and subsequent automated identification. The Glycopeptides within this library differed in glycosylation site position and glycan size ranging from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans. Factors such as glycan size, glycosylation site position within a Glycopeptide and individual precursor m/z all significantly impacted the number and quality of assignable Glycopeptide backbone fragments. Generally, high charge/low m/z precursors (>3+) and Glycopeptides carrying neutral, smaller N-glycans gave better product ion spectra, while hardly any product ions were detectable for sialylated, triply charged N-Glycopeptides. These factors impacted correct Glycopeptide identification by proteomics software tools such as SEQUEST or Amanda. A better understanding how Glycopeptide physico-chemical properties influence fragmentation will help optimizing fragmentation conditions and generate better data, which will facilitate software assisted Glycopeptide data analyses.
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glycan size and attachment site location affect electron transfer dissociation etd fragmentation and automated Glycopeptide identification
bioRxiv, 2019Co-Authors: Kathirvel Alagesan, Hannes Hinneburg, Peter H Seeberger, Daniel Varon Silva, Daniel KolarichAbstract:We used a small synthetic Glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of ETD MS/MS fragmentation and subsequent automated identification. Understanding how the physico-chemical properties of Glycopeptides influence Glycopeptide fragmentation allows for optimizing fragmentation conditions and software-assisted data analyses, which rely on informative fragmentation spectra for subsequent data processing to identify Glycopeptides. Often, mis-assignment of Glycopeptides occurs due to uncertainties such as failure to produce sufficient peptide backbone fragment ions. Our synthetic Glycopeptide library contained Glycopeptides differing in glycosylation site position within the peptide as well as glycan size (from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans). Different software solutions such as SEQUEST and Amanda were compared for ETD Glycopeptide identification. We found that all, glycan size, glycosylation site position within a Glycopeptide and individual precursor m/z significantly impacted the number and quality of assignable Glycopeptide backbone fragments, and thus the likelihood to be correctly identified in software assisted data analyses.
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it is all about the solvent on the importance of the mobile phase for zic hilic Glycopeptide enrichment
Analytical and Bioanalytical Chemistry, 2017Co-Authors: Kathirvel Alagesan, Sana Khan Khilji, Daniel KolarichAbstract:Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on Glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed “Drop-HILIC”, which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) Glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic Glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC Glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct Glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic Glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic Glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic Glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC Glycopeptide enrichment and need to be carefully considered when employed for Glycopeptide enrichment.
Jonas Nilsson - One of the best experts on this subject based on the ideXlab platform.
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Liquid chromatography-tandem mass spectrometry-based fragmentation analysis of Glycopeptides
Glycoconjugate Journal, 2016Co-Authors: Jonas NilssonAbstract:The use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS^n) for the glycoproteomic characterization of Glycopeptides is a growing field of research. The N- and O-glycosylated peptides (N- and O-Glycopeptides) analyzed typically originate from protease-digested glycoproteins where many of them are expected to be biomedically important. Examples of LC-MS^2 and MS^3 fragmentation strategies used to pursue glycan structure, peptide identity and attachment-site identification analyses of Glycopeptides are described in this review. MS^2 spectra, using the CID and HCD fragmentation techniques of a complex biantennary N-Glycopeptide and a core 1 O-Glycopeptide, representing two examples of commonly studied Glycopeptide types, are presented. A few practical tips for accomplishing Glycopeptide analysis using reversed-phase LC-MS^n shotgun proteomics settings, together with references to the latest glycoproteomic studies, are presented.
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assignment of saccharide identities through analysis of oxonium ion fragmentation profiles in lc ms ms of Glycopeptides
Journal of Proteome Research, 2014Co-Authors: Adnan Halim, Manuel Schorlemer, Christian Pett, Ulrika Westerlind, Ulla Ruetschi, Gunnar Brinkmalm, Johan Lengqvist, Göran Larson, Carina Sihlbom, Jonas NilssonAbstract:Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure–function relationships is important to better understand these events. However, characterization of glycan and Glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography–tandem mass spectrometry (LC–MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-Glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between Glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such Glycopeptides may thus be use...
Hannes Hinneburg - One of the best experts on this subject based on the ideXlab platform.
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glycan size and attachment site location affect electron transfer dissociation etd fragmentation and automated Glycopeptide identification
Glycoconjugate Journal, 2019Co-Authors: Kathirvel Alagesan, Hannes Hinneburg, Peter H Seeberger, Daniel Varon Silva, Daniel KolarichAbstract:We established a small synthetic N-Glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of electron transfer dissociation (ETD) fragmentation and subsequent automated identification. The Glycopeptides within this library differed in glycosylation site position and glycan size ranging from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans. Factors such as glycan size, glycosylation site position within a Glycopeptide and individual precursor m/z all significantly impacted the number and quality of assignable Glycopeptide backbone fragments. Generally, high charge/low m/z precursors (>3+) and Glycopeptides carrying neutral, smaller N-glycans gave better product ion spectra, while hardly any product ions were detectable for sialylated, triply charged N-Glycopeptides. These factors impacted correct Glycopeptide identification by proteomics software tools such as SEQUEST or Amanda. A better understanding how Glycopeptide physico-chemical properties influence fragmentation will help optimizing fragmentation conditions and generate better data, which will facilitate software assisted Glycopeptide data analyses.
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glycan size and attachment site location affect electron transfer dissociation etd fragmentation and automated Glycopeptide identification
bioRxiv, 2019Co-Authors: Kathirvel Alagesan, Hannes Hinneburg, Peter H Seeberger, Daniel Varon Silva, Daniel KolarichAbstract:We used a small synthetic Glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of ETD MS/MS fragmentation and subsequent automated identification. Understanding how the physico-chemical properties of Glycopeptides influence Glycopeptide fragmentation allows for optimizing fragmentation conditions and software-assisted data analyses, which rely on informative fragmentation spectra for subsequent data processing to identify Glycopeptides. Often, mis-assignment of Glycopeptides occurs due to uncertainties such as failure to produce sufficient peptide backbone fragment ions. Our synthetic Glycopeptide library contained Glycopeptides differing in glycosylation site position within the peptide as well as glycan size (from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans). Different software solutions such as SEQUEST and Amanda were compared for ETD Glycopeptide identification. We found that all, glycan size, glycosylation site position within a Glycopeptide and individual precursor m/z significantly impacted the number and quality of assignable Glycopeptide backbone fragments, and thus the likelihood to be correctly identified in software assisted data analyses.
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the art of destruction optimizing collision energies in quadrupole time of flight q tof instruments for Glycopeptide based glycoproteomics
Journal of the American Society for Mass Spectrometry, 2016Co-Authors: Hannes Hinneburg, Manfred Wuhrer, Peter H Seeberger, Daniel Varon Silva, Kathrin Stavenhagen, Ulrike Schweigerhufnagel, Stuart Pengelley, Wolfgang Jabs, Daniel KolarichAbstract:In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-Glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including Glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-Glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of Glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for Glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted Glycopeptide assignment of both N- and O-Glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-Glycopeptides and four out of five O-Glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based Glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.
Manfred Wuhrer - One of the best experts on this subject based on the ideXlab platform.
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the art of destruction optimizing collision energies in quadrupole time of flight q tof instruments for Glycopeptide based glycoproteomics
Journal of the American Society for Mass Spectrometry, 2016Co-Authors: Hannes Hinneburg, Manfred Wuhrer, Peter H Seeberger, Daniel Varon Silva, Kathrin Stavenhagen, Ulrike Schweigerhufnagel, Stuart Pengelley, Wolfgang Jabs, Daniel KolarichAbstract:In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-Glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including Glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-Glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of Glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for Glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted Glycopeptide assignment of both N- and O-Glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-Glycopeptides and four out of five O-Glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based Glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.
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glycoproteomics based on tandem mass spectrometry of Glycopeptides
Journal of Chromatography B, 2007Co-Authors: Manfred Wuhrer, Isabel M Catalina, André M. Deelder, Cornelis H HokkeAbstract:Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the Glycopeptide level. Enrichment and separation techniques for glycoproteins and Glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of Glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of Glycopeptides are presented and the following fragmentation techniques in Glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of Glycopeptides as a tool in structural glycoproteomics.