Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Summary

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive β-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Abstract

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Introduction

Glycosylation is an essential protein post-translational modification, contributing to organismal physiology, tissue pathology, and cellular recognition ^1-3. Despite major advances in analytical glycoscience, characterizing the complete diversity of glycans on a specific protein remains an extremely challenging task, especially on proteins isolated from primary biological sources. Nonetheless, the microheterogeneity of glycoprotein glycans frequently affects functional interactions with other proteins. Therefore, characterization of glycan diversity is essential for understanding the physiological significance of cellular and tissue glycosylation ^4,5

Protocol

NOTE: Lab Safety Concerns

In keeping with standard laboratory best practices, observe the following. Store all organic solvents in appropriate locations. Keep all waste materials in chemical waste containers with clear labeling of chemical compositions. As several reagents used in these protocols are potential carcinogens or generate volatile combustible gases, handle all reagents in a fume hood with ventilation. Wear personal protective equipment such as gloves, lab coat, and eye protection when working with organic solvents.

Results

Effect of Ethyl Acetate Treatment Prior to In-gel Reductive β-Elimination

A representative mass spectrum of permethylated O-linked glycan samples released from bovine mucin using in-gel reductive β-elimination is shown in Figure 3. The EtOAc wash of the gel pieces effectively removes SDS and polyacryl contaminants which interfere with subsequent MS analysis ²⁷.

Discussion

Combining in-gel reductive β-elimination with aqueous-organic extraction enhances the sensitivity and depth of structural data that can be acquired for characterizing sulfated and non-sulfated O-linked glycans harvested from small amounts of mucin-type glycoproteins resolved from other proteins by SDS-PAGE. The essential advances of the technical approaches presented in this study are: (a) facile removal of gel derived contaminants by simple washing steps; (b) quantitative recovery of permethylated sulfoglycans in t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Water, deionized water	Sigma-Aldrich	270733-4L	CHROMASOLV, for HPLC
Acetic acid, Glacial	Fisher	A38-500	Certified ACS ≥99.7% w/w
Methanol	Sigma-Aldrich	34860-4L-R	CHROMASOLV, for HPLC, ≥99.9%
Ammonium bicarbonate	Fluka	09830-500G	BioUltra, ≥99.5% (T), Step 1
Acetonitrile	Sigma-Aldrich	34998-4L	CHROMASOLV Plus, for HPLC, ≥99.9%, Step 1
Ethyl acetate	Fluka	34972-1L-R	LC-MS CHROMASOLV, Step 1
Sodium borohydride	Aldrich	213462-25G	ReagentPlus, 99%, Step 2
Iodomethane	Sigma-Aldrich	289566-100G	ReagentPlus, 99.5%,  Step 6.  Store at 4 °C until use.  Sit at room temperature before use.
Sodium hydroxide solution, 50% w/w	Fisher	SS254-1	Certified, Step 6
Dimethyl sulfoxide, anhydrous	Sigma-Aldrich	276855-1L	Anhydrous, ≥99.9%, Step 6
Methanol, anhydrous	Sigma-Aldrich	322415-100ML	Anhydrous, 99.8%, Step 6
Dichloromethane	Sigma-Aldrich	34856-4L	CHROMASOL®, for HPLC, ≥99.8%, contains amylene as stabilizer, Step 7
Dowex 50WX8 hydrogen formhydrogen form 100-200 mesh	Sigma-Aldrich	217506-500G	Step 3
AG 50W-X8 Resin	Bio-Rad	142-1441	Step 3
BAKERBOND spe 1 ml x 100 mg Solid Phase Extraction Column, PP, Octadecyl (C18) Reverse Phase	JT Baker	JT-7020-01	Step 5 and 8
7.5% Mini-PROTEAN TGX Precast Gel	Bio-Rad		Step 1
Bio-Safe Coomassie Stain	Bio-Rad	161-0786	G-250, Step 1
Silver Stain Kit for Mass Spectrometry	Pierce	24600	Step 1
Oligosaccharides Kit (Maltotriose, Dp3: R474140)	Supelco	47265
Oligosaccharides Kit (Maltotetraose, Dp4: R474135)	Supelco	47265
Disposable Pasteur Pipets, Glass, Short Tip	VWR	14673-010	Wash before use
PYREX 13 x 100 mm Disposable Round Bottom Threaded Culture Tubes	Corning	99447-13	Wash before use
PYREX 16 x 125 mm Disposable Round Bottom Threaded Culture Tubes	Corning	99447-16	Wash before use
Phenolic Caps/Closures with PTFE-Faced Rubber Liner	Kimble	45066C-13	Wash before use
Phenolic Caps/Closures with PTFE-Faced Rubber Liner	Kimble	45066C-15	Wash before use
Hamilton HPLC syringe	HAMILTON	81265	volume 500 μl, needle size 22 G
Hamilton HPLC syringe	HAMILTON	81165	volume 250 μl, needle size 22 G
Hamilton HPLC syringe	HAMILTON	81065	volume 100 μl, needle size 22s G
Hamilton HPLC syringe	HAMILTON	80965	volume 50 μl, needle size 22s G
Hamilton Calibrated Syringes	HAMILTON	80300	volume 10 μl, needle size 26s G
Petri Dish Glass 100 mm x 15mm	GSC INTERNATIONAL INC	1500-4	Wash before use, Step 1
Bard-Parker Surgical Blades	Fisher	371310	Step 1
Reacti-Therm Heating/Stirring Module	Pierce	18870	Step 1, 4 and 7
Heating Blocks	Fisher	125D	Step 2
Pyrex fiber glass wool borosilicate pore size 8 μm	Aldrich	CLS3950	Step 3
Fused Silica CutterTubing cutter	alltech	3194	Step 3
Multi-tube vortexers	VWR	444-7063	Step 6
Lyophilizer 25EL Freezemobile	Virtis	25EL
Centrifuge	VWR	Clinical 50
LTQ Orbitrap Discovery	Thermo Fisher Scientific