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 ....

Protocol

1. Culture of C. jejuni 11168H and extraction of total proteins

NOTE: C. jejuni 11168H is a hypermotile clonal derivative of NCTC 11168¹².

1. Start the cell culture by rehydrating the cell pellet (approximately 0.15 g, NCTC) with approximately 5 mL of Mueller-Hinton culture medium. Use several drops of the primary broth tube to inoculate a Mueller-Hinton agar plate. Incubate at 37 °C for 18 h under a microaerophilic atmosphere, i.e., 5% O₂, 10% CO₂, and 85% N₂.
2. Harvest bacterial lawns with 1 mL of Brain Heart Infusion (BHI) br....

Results

The method based on the combination of pronase E digestion and permethylation was applied to N-glycan analysis from total protein extracts of C. jejuni 11168H. Figure 1 shows a flowchart of the experimental procedure. In typical digestion, the ratio of enzyme to glycoprotein was set between 1:100 to 1:20. Here, the ratio of Pronase E to protein was increased to 2:1-3:1 and longer digestion was employed to obtain exhaust digestion by incubation at 37 °C for 48 h. The Asn-glycans from.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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