LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Summary

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

Abstract

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples. LERLIC-MS/MS is, therefore, the first shotgun proteomics strategy for the separation and quantification of Gln deamidation isoforms. We also demonstrate, as a novelty, that the sample processing protocol outlined here stabilizes the succinimide intermediate allowing its characterization by LERLIC-MS/MS. Application of LERLIC-MS/MS as shown in this video article can help to elucidate the currently unknown molecular arrays of protein deamidation. Additionally, LERLIC-MS/MS provides further understanding of the enzymatic reactions that encompass deamidation in distinct biological backgrounds.

Introduction

Deamidation is a protein posttranslational modification (PTM) that introduces a negative charge to the protein backbone through modification of asparagine (Asn) and/or glutamine (Gln) residues¹. This modification while affecting Asn residues generates the isomeric products isoaspartic acid (isoAsp) and n-aspartic acid (Asp) at a common 3:1 ratio². Notwithstanding, this ratio can be altered by the intervention of the repairing enzyme L-isoaspartyl methyltransferase (PIMT)^3,4. Similarly, deamidation of Gln residues generates the isomeric gamma-glutamic aci....

Protocol

1. Packing the Long-length Anion-exchange (LAX) Capillary Column

(Note: Although the LAX column can be in-home packed as we describe in this protocol, LAX columns are also commercially available, see Table of Materials and Reagents for further details).

1. Suspend 50 mg of weak anion exchange packing material in 3.5 mL of packing buffer (Table 1) to prepare the slurry.
2. Assemble the end of the capillary column (50 cm length - 200 µm internal diameter (ID) tubing) using a female-to-female fitting, a ferule and a female nut. Place a screen 1/16" OD of 1 micron pore size ins....

Results

Deamidation of Gln and Asn residues is considered a degenerative protein modification (DPM) implicated in several chronic and fatal diseases¹⁴. It has been demonstrated that this PTM can predict the half-life and degradative states of antibodies and other molecules in the human body and similar biological backgrounds^1,15. The significance of protein deamidation, in fact, goes beyond the biomedical context, .......

Discussion

In this video-article we present a step-by-step protocol of LERLIC-MS/MS³, a method to perform in-depth characterization and to accurately determine the extent of protein deamidation and the enzymatic processes involved on this protein modification. LERLIC-MS/MS is based on the use of a long-length (50 cm) LAX under the principle of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)²⁷. The use of a long-length column, as shown in our study

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PolyCAT 3 µm 100-Å (bulk material)	PolyLC Inc.	Special order
Long-length ion exchange capillary column 50 cm - 200 µm ID	PolyLC Inc.	Special order
PEEKsil Tubing 1/16" OD x 200 µm ID x 50 cm length	SGE Analytical Science under Trajan Scientific Australia	620050
Female-to-female fitting for 1/16" OD tubbing	Upchurch Scientific	UPCHF-125
Female nut for microferule	Upchurch Scientific	UPCHP-416
Microferule	Upchurch Scientific	UPCHF-132
Pressure Bomb NanoBaume	Western Fluids Engineering	SP-400
Shimadzu Prominence UFLC system	Shimadzu	Prominence UFLC
Bullet Blender	Next Advance	BBX24
Safe-lock tubes	Eppendorf	T9661-1000EA
Stainless steel beads. 0.9 – 2.0 mm. 1 lb. Non-sterile.	Next Advance	SSB14B
Table-top centrifuge	Hettich Zentrifugen	Rotina 380 R
Standard Digital Heated Circulating Bath, 120VAC	PolyScience 8006 6L	8006A11B
Sep-pack c18 desalting cartridge 50 mg	Waters	WAT020805
Vacumm concentrator	Eppendorf	Concentrator Plus System
Dionex UltiMate 3000 UHPLC	Dionex	UltiMate 3000 UHPLC
Orbitrap Elite mass spectrometer	Thermo Fisher Scientific Inc.	ORBITRAP ELITE
Michrom Thermo CaptiveSpray	Michrom-Bruker Inc.	TCSI-SS2
Incubator INCUCELL	MMM Group	INCUCELL111
Sequencing-grade modified trypsin	Promega	V5111
Protease inhibitor cocktail tablets	Roche	11836170001 (ROCHE)
Phosphate buffer solution 10x (diluted to 1x)	Sigma-Aldrich	P5493
Ammonium acetate	Sigma-Aldrich	A1542
Sodium deoxycholate	Sigma-Aldrich	D6750
Dithiothreitol	Sigma-Aldrich	D0632
Iodoacetamide	Sigma-Aldrich	i6125
Formic acid	Sigma-Aldrich	F0507 (HONEYWELL)
Ammonium hydroxide	Sigma-Aldrich	338818 (HONEYWELL)
Acetonitrile HPLC grade	Sigma-Aldrich	675415
Isopropanol HPLC grade	Sigma-Aldrich	675431
Water HPLC grade	Sigma-Aldrich	14263