Automated Assignments of N- and O-Site Specific Glycosylation with Extensive Glycan Heterogeneity of Glycoprotein Mixtures (original) (raw)
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Analytical Chemistry, 2013
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.
Molecular & Cellular Proteomics, 2013
Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either Nglycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of Nglycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed deN -glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including deN -glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their non-sugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, protein quantification based exclusively upon deglycosylated peptides may lead to inaccurate quantification.
Site-Specific Glycan-Peptide Analysis for Determination of N -Glycoproteome Heterogeneity
Journal of Proteome Research, 2013
A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography−tandem mass spectrometry (PGC-LC−MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove Nglycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the Nglycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
Site-specific protein glycosylation analysis with glycan isomer differentiation
Analytical and Bioanalytical Chemistry, 2011
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.
Towards automation of glycomic profiling of complex biological materials
Glycoconjugate Journal, 2018
Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.
Confident Assignment of Site-Specific Glycosylation in Complex Glycoproteins in a Single Step
A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC-MS and bottom-up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins. We present an LC-MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step. This system uses an online HILIC enrichment trap prior to reversed-phase C18-MS analysis. We demonstrated the effectiveness of the system using a set of glycoproteins including human transferrin (2 sequons), human alpha-1-acid glycoprotein (5 sequons), and influenza A virus hemagglutinin (9 sequons). The online enrichment renders glycopeptides the most abundant ions detected, thereby facilitating the generation of high-quality data-dependent tandem mass spectra. The tandem mass spectra exhibited product ions from both glycan and peptide backbone dissociation for a majority of the glycopeptides tested using collisionally activated dissociation that served to confidently assign site-specific glycosylation. We demonstrated the value of our system to define site-specific glycosylation using a hemagglutinin containing 9 N-glycosylation sequons from a single HILIC-C18-MS acquisition.
Simultaneous and Extensive Site-specific N- and O-Glycosylation Analysis in Protein Mixtures
Journal of Proteome Research, 2011
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.
Journal of Proteome Research, 2010
The assignment of protein glycosylation sites and their microheterogeneities are of biological importance, yet such characterization is still considered to be analytically very challenging. Several approaches have been recently developed to improve the characterization of glycosylation sites of proteins, including lectin and HILIC enrichment-based methods coupled to mass spectrometry. However, unequivocal assignment of protein glycosylation remains to be a daunting task, prompting continuous demands for the development of sensitive and cutting-edge analytical approaches.-N-Acetylglucosaminidase (endo-GlcNAc-ases, Endo-M) is an endoglycosidase capable of hydrolyzing N,N′-diacetylchitobiose moiety in N-linked oligosaccharides bound to the asparagine amino acid residue in various glycoproteins. An attractive feature of this enzyme is its ability to cleave the N,N′-diacetylchitobiose moiety while leaving an N-acetylglucosamine residue bound to the protein. This enzyme is also known to be inactive in the presence of core fucose residue linked to the reducing-end N-acetylglucosamine residue (GlcNAc). Here, we describe an approach capitalizing on these features of Endo-M to (a) determine the glycosylation sites of proteins and the occupancy of these sites, and (b) determine the attachment sites of fucose residue containing N-glycans. The latter is important because of its biological implications. Tryptically digested glycoproteins, which were subjected to Endo-M treatment, were analyzed by LC-MS/MS. Systematic evaluation of the activity of Endo-M toward different glycan structures indicated a dependence of enzyme activity on the complexity of the glycan structures. Efficient release of N-glycans using Endo-M is only achieved through the inclusion of a battery of exoglycosidases to reduce the complexity of the attached glycans and subsequently prompt an effective enzymatic release. Upon Endo-M/ exoglycosidase treatment of tryptically digested glycoproteins, glycosylated sites retain GlcNAc residue. The resulting peptides with GlcNAc residues attached to the glycosylation sites are easily assigned through LC-MS/MS analysis and subsequent database searching of the generated tandem MS of such entities. Comparing the LC-MS/MS results of the tryptic digest of glycoproteins treated with PNGase F and Endo-M/exoglycosidases allowed the assignment of core fucose residues to N-glycan reducing-ends. The detection of glycosylation sites only in the tryptic digest of PNGase F treated samples suggested core fucosylation of the attached N-glycans to such sites. This strategy was initially validated using model glycoproteins. It also proved to be useful in determining the glycosylation sites of blood serum glycoproteins.
Use of a lectin affinity selector in the search for unusual glycosylation in proteomics
Journal of Chromatography B, 2002
The purpose of the work described in this paper was to develop a new approach to the identification of glycoprotein with particular types of glycosylation. The paper demonstrates N-glycosylation sites in a glycoproteins can be identified by (1) proteolysis with trypsin, (2) lectin affinity selection, (3) enzymatic deglycosylation with peptide-N-glycosidase F (PNGase 18 F) in buffer containing 95% H O, which generates deglycosylated peptide pairs separated by 2 or 4 amu, (4) reversed-phase 2 separation of the peptide mixture and MALDI mass analysis, (5) MS-MS sequencing of the ion pairs, and (6) identification of the parent protein through a database search. This process has been tested on the selection of glycopeptides from lactoferrin and mammaglobin, and the identification of the ion pairs of fetuin glycopeptides. Glycosylation sites were 18 identified through PNGase hydrolysis in H O. During the process of hydrolyzing the conjugate, Asn is converted to an 2 18 18 aspartate residue with the incorporation of O. However, PNGase F was observed to incorporate two O into the b-carboxyl groups of the Asp residue. This suggests that the hydrolysis is at least partially reversible.