Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Summary

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Abstract

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine ('K-ε-diglycine' or simply 'diGly') can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

Introduction

The conjugation of ubiquitin to proteins marks them for degradation by the proteasome and is a crucial process in proteostasis. The C-terminal carboxyl group of ubiquitin forms an isopeptide bond with the lysine ε-amino group of the target protein^1,2. In addition, ubiquitin can be attached to other ubiquitin modules, resulting in the formation of homogeneous (i.e., K48 or K11) or branched (i.e., heterogeneous or mixed) polyubiquitin structures^1,3. The most well-known function of ubiquitin is its role in proteasomal degradation, mediated by K48-linked polyubiquitin. However, it has become clear that both mono- as well as polyubiquitination also play roles in many processes that are independent of degradation by the proteasome. For instance, K63-linked chains have nondegradative roles in intracellular trafficking, lysosomal degradation, kinase signaling, and the DNA damage response^4,5. The other six linkage types are less abundant and their roles are still largely enigmatic, although first indications about their functions in the cell are emerging, largely because of the development of novel tools to enable linkage-specific detection^6,7.

Mass spectrometry has become an indispensable tool for proteome analyses and nowadays thousands of different proteins from virtually any biological source can be identified in a single experiment. An additional layer of complexity is presented by posttranslational modifications (PTMs) of proteins (e.g., phosphorylation, methylation, acetylation, and ubiquitination) which can modulate protein activity. Large-scale identification of PTM-bearing proteins has also been made possible by developments in the mass spectrometry field. The relatively low stoichiometry of peptides bearing PTMs compared to their unmodified counterparts presents a technical challenge and biochemical enrichment steps are generally necessary prior to the mass spectrometry analysis. Over the past two decades, several different specific enrichment methods have been developed for the analysis of PTMs.

Because of the multifaceted roles of protein ubiquitination in the cell, there is a great demand for the development of analytical methods for the detection of ubiquitination sites on proteins⁸. The application of mass spectrometric methods has led to an explosion of the number of identified ubiquitination sites in fruit fly, mouse, human, and yeast proteins^{9,10,11,12,13,14}. A major step was presented by the development of immunoprecipitation based enrichment strategies at the peptide level using antibodies directed against the K-ε-GG remnant motif (also referred to as 'diglycine' or 'diGly'). These diGly peptides are produced upon digestion of ubiquitinated proteins using trypsin as the protease^15,16.

Here, we present an optimized workflow to enrich for diGly peptides using immunopurification and subsequent detection by Orbitrap mass spectrometry. Using a combination of several modifications of existing workflows, especially in the sample preparation and mass spectrometry stages, we can now routinely identify more than 23,000 diGly peptides from a single sample of HeLa cells treated with a proteasome inhibitor and ~10,000 from untreated HeLa cells. We have applied this protocol to lysates from both unlabeled and stable isotope labeling with amino acids in cell culture (SILAC) labeled HeLa cells as well as to endogenous samples such as brain tissue.

This workflow presents a valuable addition to the repertoire of tools for the analysis of ubiquitination sites in order to uncover the deep ubiquitinome. The following protocol describes all steps of the workflow in detail.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (EDC) of Erasmus MC.

1. Sample preparation

1. Cultured cells
   1. Select a cell line of interest (e.g., HeLa or osteosarcoma [U2OS] cells) and grow the cells in Dulbecco's Minimal Eagle Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin.
   2. For quantitative proteomics experiments, culture cells in DMEM lacking arginine and lysine. The medium must be supplemented with 10% dialyzed fetal bovine serum (FBS), 100 units/mL penicillin/streptomycin, and alanine-glutamine. Make two types of media by adding either conventional lysine and arginine ('Light' Medium) or lysine-8 (¹³C₆;¹⁵N₂) and arginine-10 (¹³C₆;¹⁵N₄) ('Heavy' Medium), respectively.
   3. Culture batches of cells in Light Medium (i.e., unlabeled) and Heavy Medium (i.e., labeled, SILAC) for at least six doublings before expansion and treatment to make sure that all proteins in the Heavy Medium culture are labeled with heavy stable isotope containing amino acids.
   4. Treat the cells for 8 h with 10 µM of the proteasome inhibitor bortezomib or an equivalent volume of DMSO as a mock treatment. Wash the cells with PBS, dissociate them using 1% trypsin/EDTA, and pellet the cells.
   5. Lyse the cell pellet from one 150 cm² culture plate per condition tested in 2 mL of ice-cold 50 mM Tris-HCl (pH = 8.2) with 0.5% sodium deoxycholate (DOC). Boil the lysate at 95 °C for 5 min and sonicate for 10 min (setting "H" for the sonicator listed in the Table of Materials) at 4 °C. We do not recommend the use of deubiquitinase inhibitors such as N-ethylmaleimide (NEM), because this may introduce unwanted protein modifications that can complicate peptide identification.
2. In vivo mouse brain tissue
   1. When using in vivo tissue, lyse the tissue in an ice-cold buffer containing 100 mM Tris-HCl (pH = 8.5), 12 mM sodium DOC, and 12 mM sodium N-lauroylsarcosinate¹⁷. Sonicate the lysate for 10 min (setting "H" for the sonicator listed in the Table of Materials) at 4 °C and boil the lysate for 5 min at 95 °C.
3. Quantitate the total protein amount using a colorimetric absorbance BCA protein assay kit. The total amount of protein should be at least several milligrams for a successful diGly peptide immunoprecipitation (IP). For SILAC experiments mix the light and heavy labeled proteins in a 1:1 ratio based on the total protein amount.
4. Reduce all proteins using 5 mM 1,4-dithiothreitol for 30 min at 50 °C and subsequently alkylate them with 10 mM iodoacetamide for 15 min in the dark. Perform protein digestion with Lys-C (1:200 enzyme-to-substrate ratio) for 4 h followed by overnight digestion with trypsin (1:50 enzyme-to-substrate ratio) at 30 °C or at room temperature (RT).
5. Add trifluoroacetic acid (TFA) to the digested sample to a final concentration of 0.5% and centrifuge the sample at 10,000 x g for 10 min in order to precipitate and remove all detergent. Collect the supernatant containing the peptides for subsequent fractionation.

2. Offline peptide fractionation

1. Use high pH reverse-phase (RP) C18 chromatography with polymeric stationary phase material (300 Å, 50 µM; see Table of Materials) loaded into an empty column cartridge to fractionate the tryptic peptides. The stationary phase bed size must be adjusted to the amount of protein digest to be fractionated. Prepare an empty 6 mL column cartridge (see Table of Materials) filled with 0.5 g of stationary phase material for ~10 mg of protein digest. The protein digest to stationary phase ratio should be approximately 1:50 (w/w).
2. Load the peptides onto the prepared column and wash the column with approximately 10 column volumes of 0.1% TFA, followed by approximately 10 column volumes of H₂O.
3. Elute the peptides into three fractions with 10 column volumes of 10 mM ammonium formate solution (pH = 10) with 7%, 13.5%, and 50% acetonitrile (AcN), respectively. Lyophilize all fractions to completeness.
4. Use ubiquitin remnant motif (K-ε-GG) antibodies conjugated to protein A agarose beads for the immunoenrichment of diGly peptides. Because the exact amount of antibody per batch of beads is proprietary information and not disclosed by the manufacturer, it is recommended to use the same definition for a batch of beads as the manufacturer does in order to avoid confusion. Wash one batch of these beads 2x with PBS and split the bead slurry into six equal fractions. See Figure 1 for a detailed experimental scheme.
5. Dissolve the three peptide fractions collected in step 2.3 in 1.4 mL of a buffer composed of 50 mM MOPS, 10 mM sodium phosphate, and 50 mM NaCl (pH = 7.2), and spin down the debris.
6. Add the supernatants of the fractions to the diGly antibody beads and incubate for 2 h at 4 °C on a rotator unit. Spin down the beads and transfer the supernatant to a fresh batch of antibody beads and incubate again for 2 h at 4 °C.
7. Store the supernatants for subsequent global proteome (GP) analysis.
8. Transfer the beads from every fraction into a 200 µL pipette tip equipped with a GF/F filter plug to retain the beads. Put the pipette tip with the beads into a 1.5 mL microcentrifuge tube equipped with a centrifuge tip adapter. Wash the beads 3x with 200 µL of ice-cold IAP buffer and subsequently 3x with 200 µL of ice-cold milliQ H₂O. Spin down the column at 200 x g for 2 min before every wash step, but be careful not to let the column run dry. Elute the peptides using 2 cycles of 50 µL of 0.15% TFA.
9. Desalt the peptides using a C18 stage tip (essentially a 200 µL pipette tip with two C18 disks) and dry them to completeness using vacuum centrifugation.

3. Nanoflow LC-MS/MS

1. Perform LC-MS/MS experiments on a sensitive mass spectrometer coupled to a nanoflow LC system.
2. Use an in-house packed 50 cm reversed-phase column with a 75 µm inner diameter packed with CSH130 resin (3.5 µm, 130 Å) and elute the peptides with a gradient of 2−28% (AcN, 0.1% FA) over 120 min at 300 nL/min. Alternatively, use commercially available LC columns with similar properties. Keep the column at 50 °C using a column oven, for example (see Table of Materials).
3. Perform the mass spectrometry analysis.
   1. The mass spectrometer must be operated in data-dependent acquisition (DDA) mode. MS1 mass spectra should be collected at high resolution (e.g., 120,000), with an automated gain control (AGC) target setting of 4E5 and a maximum injection time of 50 ms in case of an Orbitrap mass spectrometer.
   2. Perform the mass spectrometry analysis in "Highest Intensity First" mode first. This way, the most intense ion is selected first for fragmentation, then the second highest, and so forth, using the top speed method with a total cycle time of 3 s. Subsequently, perform a second round of DDA MS analysis in "Lowest Intensity First" mode, so that the least intense ion is selected first, then the second lowest, and so forth. This strategy ensures optimal detection of very low abundancy peptides.
   3. Filter the precursor ions according to their charge states (2-7 charges) and monoisotopic peak assignment. Exclude previously interrogated precursors dynamically for 60 s. Isolate peptide precursors with a quadrupole mass filter set to a width of 1.6 Th.
   4. Collect MS2 spectra in the ion trap at an AGC of 7E3 with a maximum injection time of 50 ms and HCD collision energy of 30%.

4. Data analysis

1. Analyze the mass spectrometry raw files using an appropriate search engine such as the freely available MaxQuant software suite based on the Andromeda search engine^18,19. In MaxQuant, select the default settings with a few adaptations that are indicated below. Set the enzyme specificity to trypsin, with the maximum number of missed cleavages raised to three. Set lysine with a diGly remnant (+114.04 Da), oxidation of methionine and N-terminal acetylation as variable modifications, and set carbamidomethylation of cysteine as a fixed modification.
2. Perform database searches against a FASTA file containing protein sequences downloaded from, for example, the Uniprot repository (https://www.uniprot.org/downloads) combined with a decoy and a standard common contaminant database that is automatically provided by MaxQuant. Set the false discovery rate (FDR) to 1% and set the minimum score for modified (diGly) peptides to 40 (default value). Exclude peptides identified with a C-terminal diGly modified lysine residue from further analysis.
3. For the quantitative analysis of SILAC experiment files, set the multiplicity to "2" and repeat step 4.2.
4. Perform all downstream analyses (e.g., statistics, gene ontology analyses) with the Perseus module²⁰ of the MaxQuant software suite.

Results

Ubiquitinated proteins leave a 114.04 Da diglycine remnant on the target lysine residue when the proteins are digested with trypsin. The mass difference caused by this motif was used to unambiguously recognize the site of ubiquitination in a mass spectrometry experiment. The strategy that we describe here is a state-of-the-art method for the enrichment and subsequent identification of diGly peptides by nanoflow LC-MS/MS (Figure 1A). In this s...

Discussion

The protocol described here was applied to samples from various biological sources, such as cultured cells and in vivo tissue. In all cases we identified thousands of diGly peptides, provided that the total protein input amount was at least 1 mg. The enrichment using specific antibodies is highly efficient, given that only at most 100-150 very low abundant diGly peptides were identified from whole cell lysates if no enrichment procedures for ubiquitinated proteins or diGly peptides were applied. Obviously, sensitive mass...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,4-Dithioerythritol	Sigma-Aldrich	D8255
3M Empore C18 Octadecyl disks	Supelco	66883-U	product discontinued at Supelco; CDS Analytical is the new manufacturer (https://www.cdsanalytical.com/empore)
Ammonium formate	Sigma-Aldrich	70221
Bortezomib	UBPbio
CSH130 resin, 3.5 μm, 130 Å	Waters
Dimethylsulfoxide (DMSO)	Sigma-Aldrich	34869
DMEM	ThermoFisher
EASY-nanoLC 1200	ThermoFisher
FBS	Gibco
GF/F filter plug	Whatman	1825-021
Iodoacetamide	Sigma-Aldrich	I6125
Lysine, Arginine	Sigma-Aldrich
Lysine-8 (13C6;15N2), Arginine-10 (13C6;15N4)	Cambridge Isotope Laboratories
Lysyl Endopeptidase(LysC)	Wako Pure Chemicals	129-02541
NanoLC oven	MPI design, MS Wil GmbH
N-Lauroylsarcosine sodium salt	Sigma-Aldrich	L-5125
Orbitrap Fusion Lumos mass spectrometer	ThermoFisher
Pierce BCA Protein Assay Kit	ThermoFisher / Pierce	23225
PLRP-S (300 Å, 50 µm) polymeric reversed phase particles	Agilent Technologies	PL1412-2K01
PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit	Cell Signaling Technologies	5562
Sep-Pak tC18 6 cc Vac Cartridge	Waters	WAT036790	Remove the tC18 material from the cartridge before filling the cartridge with PLRP-S
Sodium deoxycholate	Sigma-Aldrich	30970
Tris-base	Sigma-Aldrich	T6066
Tris-HCl	Sigma-Aldrich	T5941
Trypsin, TPCK Treated	ThermoFisher	20233