Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Summary

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Abstract

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

Introduction

The close regulation of protein function and stability is of paramount importance, as they are major drivers of phenotypic control of biology. The function of a protein is constructed from two components: its intrinsic polypeptide sequence and any posttranslational modifications (PTMs). Various chemical PTMs have been identified including glycosylation, phosphorylation, acetylation, and methylation¹. In 1975, Goldstein et al.² identified a small protein and named it ubiquitin due to its ubiquitous nature. Ubiquitin was found to be important in protein degradation³. However, since then it has been ....

Protocol

1. Preparation of a heavy peptide standard

1. Depending on the supplier and quality of the heavy peptides purchased, the heavy peptides will need to be diluted. This protocol used the peptide sequences reported in Table 1, with the C–terminal amino acid modified to Lysine (¹³C₆¹⁵N₂-lysine) or Arginine (¹³C₆¹⁵N₄-arginine).
   1. Mix the heavy peptides, diluting the mix with 50% acetonitrile (ACN) to create a peptide mix with each peptide at 10 µM.
   2. Create aliquots of the peptide mix and store at -80 °C.

Results

To demonstrate the use of a ubiquitin chain analysis by PRM, three cell lines were selected: a mouse melanoma cell line B16, and the two common human cell lines A549 (adenocarcinomic alveolar basal epithelial cells) and HeLa (cervical cancer cells). These cultures grew to midexponential phase in appropriate media before being treated with 0, 10, or 100 mM MG-132 for 4 h prior to harvest. MG-132 is a proteasome inhibitor preventing the degradation of ubiquitin-conjugated proteins by the proteasome¹⁴

Discussion

Analysis of the ubiquitin state within a proteome is of increasing importance to a wide variety of biological questions. The description of the ubiquitination state of a sample must focus not only on the profile of proteins being ubiquitinated but also on the topology of such ubiquitination. The assessment of this topology by targeted MS, as described here, has a role in a wide range of biological investigations.

It should be understood that the protocol outlined here provides a global topolog.......

Materials

Name	Company	Catalog Number	Comments
Acetonitrile (ACN)	Merck	100029
Ammonium bicarbonate (ABC)	Fluka	9830
Centrifuge	Beckman Coulter	Microfuge 16
Chloroacetamide (CAA)	Sigma	22790
Eppendorf LoBind	Eppendorf	22431081
Formic acid (FA)	Thermo Fisher Scientific	85178
Heavy Peptides	JPT Peptide Technologies
HPLC	Dionex	Ulitimate 3000
LC Column	Thermo Fisher Scientific	160321
Lys C	Wako	125-05061
Mass Spectrometer	Thermo Fisher Scientific	Q-Exactive Plus
N-ethylmaleimide (NEM)	ACROS Organics	156100050
Positive Control Chain K48	Boston Biochem	UC-240
Positive Control Chain K63	Boston Biochem	UC-340-100
Positive Control Chain M1	Boston Biochem	UC-710B-025
Sodium Hydroxide (NaOH)	Sigma	S5881
Sonifier	Branson sonifier	SFX 150
Thermomixer	Eppendorf	Thermomixer Comfort
Trifluoroacetic acid (TFA)	Sigam	T6508
Tris(2-carboxyethyl)phosphine (TCEP)	Thermo Fisher Scientific	77720
Trypsin	Promega	V1511A
Urea	Sigma	51456
Waters μElution C18 plates	Waters	186002318