Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins
Summary
This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.
Abstract
Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1−3:1) and a longer incubation time (48−72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.
Introduction
Protein N-glycosylation is one of the most common and complex post-translational modifications in eukaryotes1. N-glycans play an essential role in protein folding and also have an impact on protein sorting in biosynthetic traffic2. Mass spectrometry has been widely used in the analysis of glycans released by exoglycosidase cleavage (glycomics). The remaining deglycosylated peptides (glycoproteomics) have been commonly used to identify the sequences of glycosylated peptides in eukaryotes3. The identification of N-linked glycans in Campylobacter jejuni suggested that N-glycosylation is not restricted to eukaryotes4,5,6,7,8. However, for bacteria, there is a lack of effective exoglycosidases or endoglycosidases to release oligosaccharides for glycan analysis.
An alternative approach has been developed to characterize glycosylation sites and glycan structures in glycoproteins, which is based on the use of nonspecific proteolysis to digest most peptides' backbone and generate pseudo-oligosaccharides that only contain a few amino acids. Various non-specific enzymes have been used to generate pseudo-oligosaccharides and it has been found that Pronase E offered the most efficient and reproducible digestion9. We have developed a glycomics strategy based on Pronase E to analyze C. jejuni N-glycans10,11. The pseudo-oligosaccharides were analyzed directly by capillary electrophoresis mass spectrometry (CE-MS) and/or by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after permethylation.
Here, a universal method is described for glycomics analysis that uses nonspecific Pronase E digestion and permethylation to study glycosylation from mucosal pathogen C. jejuni. This method is capable of characterizing N-linked glycans expressed by both eukaryotic and bacterial systems and is also useful in identifying novel intermediates in N-linked glycosylation pathways.
Protocol
Representative Results
Discussion
There are two critical steps in the protocol for the implementation of this universal glycomics strategy. The first critical step is the completion of exhaust digestion. This method strongly depends on the completion of Pronase E digestion. It is thus essential to use a long digestion time and a high enzyme-to-protein ratio. Typically, it is suggested to use a ratio of 2:1-3:1 for Pronase E/protein, and an incubation time of 48 h. It has been demonstrated that this proposed exhaust digestion produced the glycans with a s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Kenneth Chan, David J. McNally, Harald Nothaft, Christine M. Szymanski, Jean-Robert Brisson, and Eleonora Altman for assistance and helpful discussions.
Materials
| 2,5-dihydroxybenzoic acid (DHB), | Sigma-Aldrich (St. Louis, MO) | 149357 | |
| 2-Propanol | Fisher Scientific( Ottawa, Ontario,Canada) | AA22906K7 | |
| 4000 Q-Trap | AB Sciex (Concord, Canada) | Mass spectrometer | |
| 4800 MALDI-TOF/TOF | Applied Biosystems (Foster City, CA) | Mass spectrometer | |
| acetonitrile (ACN) | Fisher Scientific( Ottawa, Ontario,Canada) | A996-4 | |
| Brain Heart Infusion (BHI) broth | Sigma-Aldrich (St. Louis, MO) | 5121 | |
| C18 Sep-Pak cartridges | Waters (Milford, MA). | WAT036945 | |
| chloroform | Sigma-Aldrich (St. Louis, MO) | CX1054 | |
| Difco | Fisher Science (Ottawa, Ontario,Canada) | DF0037-17-8 | Fisher Science |
| dimethyl sulfoxide (DMSO) | Sigma-Aldrich (St. Louis, MO) | 276855 | |
| Eppendorf tube | Diamed (Missisauga, Ontario,Canada) | SPE155-N | |
| glacial acetic acid | Sigma-Aldrich (St. Louis, MO) | A6283 | |
| methanol | Fisher Scientific, Ottawa, Ontario,Canada) | A544-4 | |
| methyl iodide | Sigma-Aldrich (St. Louis, MO) | 289566 | |
| PGC cartridge | Thermo Scientific(Waltham,MA) | 60106-303 | Porous graphitic carbon |
| Pronase E | Sigma-Aldrich (St. Louis, MO) | 7433-2 | |
| Protein Assay Kit | Bio-rad (Mississauga, Ontario, Canada) | 5000001 | |
| Refrigerated vacuum concentrator | Thermo Scientific(Waltham,MA) | SRF110P2-230 | |
| sodium hydroxide | Sigma-Aldrich (St. Louis, MO) | 367176 | |
| Sonicator Ultrasonic Processor | Mandel Scientific (Guelph, Ontario,Canada) | XL 2020 | |
| Trifluoacetic acid (TFA) | Fisher Scientific( Ottawa, Ontario,Canada) | A11650 | |
| Tris-HCl | Sigma-Aldrich (St. Louis, MO) | T3253 |
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