In-Gel Nonspecific Proteolysis for Elucidating Glycoproteins: A Method for Targeted Protein-Specific Glycosylation Analysis in Complex Protein Mixtures (original) (raw)
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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.
Molecular & Cellular Proteomics, 2004
Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-ofthe-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.
Improving the Study of Protein Glycosylation with New Tools for Glycopeptide Enrichment
Fundamentals of Glycosylation [Working Title], 2021
High confidence methods are needed for determining the glycosylation profiles of complex biological samples as well as recombinant therapeutic proteins. A common glycan analysis workflow involves liberation of N-glycans from glycoproteins with PNGase F or O-glycans by hydrazinolysis prior to their analysis. This method is limited in that it does not permit determination of glycan attachment sites. Alternative proteomics-based workflows are emerging that utilize site-specific proteolysis to generate peptide mixtures followed by selective enrichment strategies to isolate glycopeptides. Methods designed for the analysis of complex samples can yield a comprehensive snapshot of individual glycans species, the site of attachment of each individual glycan and the identity of the respective protein in many cases. This chapter will highlight advancements in enzymes that digest glycoproteins into distinct fragments and new strategies to enrich specific glycopeptides.
Journal of Proteome Research, 2013
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.
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.
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.
2016
ABSTRACT: 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 automati-cally distinguishes correct assignments from random matches and complements experimental techniques that are optimal for glycopeptides, including nonspecific proteolysis and high mass resolution liquid chromatography/ tandem mass spectrometry (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 nonrandom glycopeptide matches and demonstrating the proof-of-concept for a self-consist...
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.