Charles Nwosu - Academia.edu (original) (raw)

Papers by Charles Nwosu

Glycobiology, Feb 22, 2013

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as b... more Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structurerelated specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomerspecific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.

Pda Journal of Pharmaceutical Science and Technology, 2019

The application of advanced methodologies such as next-generation sequencing (NGS) and mass spect... more The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. Currently, there is little clarity and consensus on the utilization, timing, and significance of SV findings. This white paper addresses the current practices, logistics, and strategies for the analysis of SVs using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences including approaches for detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback. Although SVs are a potential issue for all recombinant protein therapeutics, the scope of this discussion will be limited to SVs produced in mammalian cells. Ultimately, it is our hope that the findings from the survey and deliberations of the committee are useful to decision makers in industry and positions them to respond to findings of SVs in recombinant proteins that are destined for clinical or commercial use in a strategic manner. LAY ABSTRACT: This white paper addresses the current practices, logistics, and strategies for the analysis of amino acid sequence variants using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences regarding detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback.

Analytical and Bioanalytical Chemistry, Sep 22, 2022

Recombinant protein engineering design affects therapeutic properties including protein efficacy,... more Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC-MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics.

Analytical Chemistry, Dec 28, 2012

Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common... more Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem MS analysis of the resulting N-and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as pronase, proteinase K, pepsin, papain and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data and predetermined protein I.D. (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (kappa casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid deter mination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.

Analytical Chemistry, May 24, 2013

Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits fu... more Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and compliments experimental techniques that are optimal for glycopeptides, including non-specific proteolysis and high mass resolution LC/MS/ MS. SSG for multiple N-and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of non-random glycopeptide matches and demonstrating the proof-of-concept for a selfconsistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.

Molecular & Cellular Proteomics, Sep 1, 2012

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in ... more Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-␤-Nacetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man 3 GlcNAc 2 (␣1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies. Molecular &

The FASEB Journal, Apr 1, 2012

Analytical and Bioanalytical Chemistry

Recombinant protein engineering design affects therapeutic properties including protein efficacy,... more Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC–MS methods, the different glycoforms at a given...

Annotation and structural elucidation of bovine milk

PDA Journal of Pharmaceutical Science and Technology, 2019

The application of advanced methodologies such as next-generation sequencing (NGS) and mass spect... more The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. Currently, there is little clarity and consensus on the utilization, timing, and significance of SV findings. This white paper addresses the current practices, logistics, and strategies for the analysis of SVs using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences including approaches for detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback. Although SVs are a potential issue for all recombinant protein therapeutics, the scope of this discussion will be limited to SVs produced in mammalian cells. Ultimately, it is our hope that the findings from the survey and deliberations of the committee are useful to decision makers in industry and positions them to respond to findings of SVs in recombinant proteins that are destined for clinical or commercial use in a strategic manner. LAY ABSTRACT: This white paper addresses the current practices, logistics, and strategies for the analysis of amino acid sequence variants using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences regarding detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback.

Molecular & Cellular Proteomics, 2012

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in ... more Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-␤-Nacetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man 3 GlcNAc 2 (␣1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies. Molecular &

Journal of Proteome Research, 2012

The isolation of whey proteins from human and bovine milks followed by profiling of their entire ... more The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.

Journal of Proteome Research, 2013

Despite recent advances, site-specific profiling of protein glycosylation remains a significant a... more Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site-and structure-specific information about protein glycosylation.

Journal of Proteome Research, 2011

Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to th... more Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining non-specific protease digestion, nano-flow liquid chromatography and tandem mass spectrometry (MS/MS) aimed at comprehensive sitespecific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein and bovine fetuin using nano-flow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan micro-heterogeneity information was obtained. This approach presents a platform to simultaneously characterize N-and O-glycosites in the same mixture with extensive site heterogeneity.

Glycobiology, 2013

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as b... more Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structurerelated specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomerspecific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.

Bioconjugate Chemistry, 2012

Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a t... more Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a target protein has many potential applications in biomedical science. For example, the properties of probes used as receptor-imaging ligands may be improved by increasing their residence time on the targeted receptor. Among the more interesting cases are peptide ligands, the strongest of which typically bind to receptors with micromolar dissociation constants, and which may depend on processes other than simple binding to provide images. The side chains of cysteine, histidine, or lysine are attractive for chemical attachment to improve binding to a receptor protein, and a system based on acryloyl probes attaching to engineered cysteine provides excellent positron emission tomographic images in animal models (Wei et al. (2008) J. Nucl. Med. 49, 1828-1835). In nature, lysine is a more common but less reactive residue than cysteine, making it an interesting challenge to modify. To seek practically useful cross-linking yields with naturally occurring lysine side chains, we have explored not only acryloyl but also other reactive linkers with different chemical properties. We employed a peptide-VEGF model system to discover that a 19mer peptide ligand, which carried a lysine-tagged dinitrofluorobenzene group, became attached stably and with good yield to a unique lysine residue on human vascular endothelial growth factor (VEGF), even in the presence of 70% fetal bovine serum. The same peptide carrying acryloyl and related Michael acceptors gave low yields of attachment to VEGF, as did the chloroacetyl peptide.

Analytical Chemistry, 2013

Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits fu... more Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and compliments experimental techniques that are optimal for glycopeptides, including non-specific proteolysis and high mass resolution LC/MS/ MS. SSG for multiple N-and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of non-random glycopeptide matches and demonstrating the proof-of-concept for a selfconsistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.

Analytical Chemistry, 2010

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with ... more A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrixassisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collisioninduced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positiveand negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved sitespecific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N-and Olinked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis. Glycosylation is one of the most ubiquitous forms of posttranslational modifications of proteins and involves the enzymatic attachment of glycans to asparagine (N-glycans) and serine or threonine (O-glycans). 1 Glycosylation has vital roles in protein folding, 2,3 protein

Analytical Chemistry, 2013

Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common... more Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem MS analysis of the resulting N-and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as pronase, proteinase K, pepsin, papain and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data and predetermined protein I.D. (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (kappa casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid deter mination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.

Analytical and Bioanalytical Chemistry, 2011

Glycosylation is one of the most common yet diverse post-translational modifications. Information... more Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one Nglycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.

Glycobiology, Feb 22, 2013

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as b... more Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structurerelated specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomerspecific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.

Pda Journal of Pharmaceutical Science and Technology, 2019

The application of advanced methodologies such as next-generation sequencing (NGS) and mass spect... more The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. Currently, there is little clarity and consensus on the utilization, timing, and significance of SV findings. This white paper addresses the current practices, logistics, and strategies for the analysis of SVs using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences including approaches for detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback. Although SVs are a potential issue for all recombinant protein therapeutics, the scope of this discussion will be limited to SVs produced in mammalian cells. Ultimately, it is our hope that the findings from the survey and deliberations of the committee are useful to decision makers in industry and positions them to respond to findings of SVs in recombinant proteins that are destined for clinical or commercial use in a strategic manner. LAY ABSTRACT: This white paper addresses the current practices, logistics, and strategies for the analysis of amino acid sequence variants using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences regarding detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback.

Analytical and Bioanalytical Chemistry, Sep 22, 2022

Recombinant protein engineering design affects therapeutic properties including protein efficacy,... more Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC-MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics.

Analytical Chemistry, Dec 28, 2012

Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common... more Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem MS analysis of the resulting N-and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as pronase, proteinase K, pepsin, papain and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data and predetermined protein I.D. (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (kappa casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid deter mination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.

Analytical Chemistry, May 24, 2013

Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits fu... more Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and compliments experimental techniques that are optimal for glycopeptides, including non-specific proteolysis and high mass resolution LC/MS/ MS. SSG for multiple N-and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of non-random glycopeptide matches and demonstrating the proof-of-concept for a selfconsistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.

Molecular & Cellular Proteomics, Sep 1, 2012

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in ... more Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-␤-Nacetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man 3 GlcNAc 2 (␣1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies. Molecular &

The FASEB Journal, Apr 1, 2012

Analytical and Bioanalytical Chemistry

Recombinant protein engineering design affects therapeutic properties including protein efficacy,... more Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC–MS methods, the different glycoforms at a given...

Annotation and structural elucidation of bovine milk

PDA Journal of Pharmaceutical Science and Technology, 2019

The application of advanced methodologies such as next-generation sequencing (NGS) and mass spect... more The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. Currently, there is little clarity and consensus on the utilization, timing, and significance of SV findings. This white paper addresses the current practices, logistics, and strategies for the analysis of SVs using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences including approaches for detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback. Although SVs are a potential issue for all recombinant protein therapeutics, the scope of this discussion will be limited to SVs produced in mammalian cells. Ultimately, it is our hope that the findings from the survey and deliberations of the committee are useful to decision makers in industry and positions them to respond to findings of SVs in recombinant proteins that are destined for clinical or commercial use in a strategic manner. LAY ABSTRACT: This white paper addresses the current practices, logistics, and strategies for the analysis of amino acid sequence variants using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences regarding detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback.

Molecular & Cellular Proteomics, 2012

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in ... more Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-␤-Nacetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man 3 GlcNAc 2 (␣1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies. Molecular &

Journal of Proteome Research, 2012

The isolation of whey proteins from human and bovine milks followed by profiling of their entire ... more The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.

Journal of Proteome Research, 2013

Despite recent advances, site-specific profiling of protein glycosylation remains a significant a... more Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site-and structure-specific information about protein glycosylation.

Journal of Proteome Research, 2011

Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to th... more Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining non-specific protease digestion, nano-flow liquid chromatography and tandem mass spectrometry (MS/MS) aimed at comprehensive sitespecific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein and bovine fetuin using nano-flow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan micro-heterogeneity information was obtained. This approach presents a platform to simultaneously characterize N-and O-glycosites in the same mixture with extensive site heterogeneity.

Glycobiology, 2013

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as b... more Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structurerelated specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomerspecific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.

Bioconjugate Chemistry, 2012

Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a t... more Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a target protein has many potential applications in biomedical science. For example, the properties of probes used as receptor-imaging ligands may be improved by increasing their residence time on the targeted receptor. Among the more interesting cases are peptide ligands, the strongest of which typically bind to receptors with micromolar dissociation constants, and which may depend on processes other than simple binding to provide images. The side chains of cysteine, histidine, or lysine are attractive for chemical attachment to improve binding to a receptor protein, and a system based on acryloyl probes attaching to engineered cysteine provides excellent positron emission tomographic images in animal models (Wei et al. (2008) J. Nucl. Med. 49, 1828-1835). In nature, lysine is a more common but less reactive residue than cysteine, making it an interesting challenge to modify. To seek practically useful cross-linking yields with naturally occurring lysine side chains, we have explored not only acryloyl but also other reactive linkers with different chemical properties. We employed a peptide-VEGF model system to discover that a 19mer peptide ligand, which carried a lysine-tagged dinitrofluorobenzene group, became attached stably and with good yield to a unique lysine residue on human vascular endothelial growth factor (VEGF), even in the presence of 70% fetal bovine serum. The same peptide carrying acryloyl and related Michael acceptors gave low yields of attachment to VEGF, as did the chloroacetyl peptide.

Analytical Chemistry, 2013

Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits fu... more Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and compliments experimental techniques that are optimal for glycopeptides, including non-specific proteolysis and high mass resolution LC/MS/ MS. SSG for multiple N-and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of non-random glycopeptide matches and demonstrating the proof-of-concept for a selfconsistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.

Analytical Chemistry, 2010

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with ... more A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrixassisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collisioninduced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positiveand negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved sitespecific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N-and Olinked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis. Glycosylation is one of the most ubiquitous forms of posttranslational modifications of proteins and involves the enzymatic attachment of glycans to asparagine (N-glycans) and serine or threonine (O-glycans). 1 Glycosylation has vital roles in protein folding, 2,3 protein

Analytical Chemistry, 2013

Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common... more Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem MS analysis of the resulting N-and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as pronase, proteinase K, pepsin, papain and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data and predetermined protein I.D. (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (kappa casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid deter mination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.

Analytical and Bioanalytical Chemistry, 2011

Glycosylation is one of the most common yet diverse post-translational modifications. Information... more Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one Nglycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.