Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Summary

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Abstract

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

Introduction

The method proposed here is dedicated to study PTMs mediated by the ubiquitin family members from cultured mammalian cells in order to identify potential alterations associated with a specific condition (treatment, differentiation, etc). PTMs represent the last step of regulation of proteins' functions¹. Indeed, once produced by the translational machinery, most if not all proteins undergo different kinds of PTMs that modulate their activity, molecular interactions, and intracellular location¹. Among the plethora of PTMs are the ones mediated by the ubiquitin family of proteins, ubiquitin itself and all ubiquitin-lik....

Protocol

1. Generation of stable cell lines expressing 6His-Flag-Ubl

NOTE: Co-transfection of HEK-293T cells with pCCL-6HF-Ubl, pVSVG and delta-Helper.

1. Day 0: Seed 293T cells in a 6-well plate to obtain 50-70% confluence the day after.
2. Day 1: Co-transfect 50-70% confluent cells with a mix of 1 µg of pCCL-6HF-Ubl or pCCL-GFP, 1 µg of pVSVG and 1 µg of delta-Helper vectors, using a transfection reagent and protocol for lentivirus production. After 6 h of transfection, change the medium to a fresh one corresponding to cells to be transduced. Seed the cells to be transduced in a 6 well plate in order to obtain a 10-20% confluence t....

Results

Transduction of culture mammalian cells to create GFP and 6HF-Ub expressing cells
To produce lentiviruses which will be used later to transduce MiaPaCa-2 cells, 70% confluent HEK-293T cells are co-transfected with an equal amount of the three vectors, pCCL-6HF-Ubiquitin or GFP/Delta-Helper/pvSvG. After 24 h of production, the medium containing lentiviral particles is recovered and filtered. It is possible at this point to control the efficiency of the transfection by .......

Discussion

We have developed a robust and reliable methodology to generate profiles of proteins modified by the main ubiquitin family members. Indeed, we have successfully applied this protocol to generate profiles of PTMs by ubiquitin, and also by SUMO and Nedd8, and to detect alterations associated with a treatment⁷, in response to the over expression or knockdown of a certain gene (data not shown) and in cells that acquired a resistant phenotype to diverse chemotherapeutic drugs.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ANTI-FLAG M2 Affinity Gel	Sigma-Aldrich	A2220-5ML	binds all Flag tagged proteins
anti-Flag M2 antibody	Sigma-Aldrich	F3165	to detect 6His-Flag tagged expression of ub/ubl
Cell strainer 40 µm	Falcon	352350	to remove floating pellet from guanidine lysed cells
Flag peptide	Sigma-Aldrich	F3290	elute flag tagged proteins from anti-flag beads
Guanidine hydrochloride	Sigma-Aldrich	50933	chaotropic agent used to denature all proteins in cell lysate
Imidazole	Sigma-Aldrich	I5513	eluates 6His bond proteins from Ni-NTA beads
Lipofectamine 3000	ThermoFisher	L3000015	to transfect HEK-293T cells to produce lentiviruses
Lobind tubes	Sigma-Aldrich	Z666491	avoids absorption of precious material
Membrane Filter, 0.45 µm	Millipore	HAWP04700F1	to filter the lentiviral supernantant
Ni-NTA	Qiagen	30210	purification of the 6His tag