© 2013 Nature America, Inc. All rights reserved.
Large-scale identification of ubiquitination sites by
1950 | VOL.8 NO.10 | 2013 | nature protocols
Protein ubiquitination is an important post-translational modi-
fication (PTM) that is essential for regulating protein turnover
through the ubiquitin-proteasome system. Ubiquitination occurs
most commonly on the ε-amino group of protein lysine residues
through the concerted action of activating (E1), conjugating (E2)
and ligating (E3) enzymes1. As ubiquitin itself has seven lysine
residues, substrates can be monoubiquitinated or polyubiquiti-
nated, resulting in various ubiquitin chain lengths and topologies
that may serve to specifically bind cellular proteins with distinct
Historically, the identification of protein ubiquitination sites
by MS has proven to be challenging because of the low stoi-
chiometry of ubiquitinated proteins, the size of the modifica-
tion itself and the diversity in resulting ubiquitin chain types.
To enhance the identification of low-abundance ubiquitinated
proteins in complex samples, earlier studies have used over-
expressed, affinity-tagged ubiquitin systems to aid in the enrich-
ment and identification of ubiquitinated proteins3,4. These
methods rely on the enrichment of ubiquitinated proteins when
ubiquitin is intact and bound to its substrate. After protein diges-
tion, both formerly ubiquitinated peptides and nonubiquitinated
peptides (from the enriched substrate proteins and the attached
ubiquitin molecules) are present in the sample. The increased
sample complexity resulting from the presence of nonubiquiti-
nated peptides makes detection of specific sites of ubiquitination
challenging. Protein-level enrichment methods have enabled the
detection of several thousand putative, ubiquitinated substrate
proteins, but they lack the necessary enrichment specificity to
enable identification of large numbers of ubiquitination sites.
Robust, large-scale detection of endogenous ubiquitination
sites by MS requires a technique that facilitates the specific enrich-
ment of only the modified lysine-containing peptides of ubiqui-
tinated substrate proteins. To this end, global analysis of protein
ubiquitination has markedly improved with the commercializa-
tion of antibodies specific for the di-glycyl remnant produced on
ubiquitinated lysine residues (K-ε-GG) after trypsin digestion5–8.
Specifically, trypsin digestion of ubiquitinated proteins cleaves off
all but the two C-terminal glycine residues of ubiquitin from the
modified protein. These two C-terminal glycine (GG) residues
remain linked to the ε-amino group of the modified lysine resi-
due in the tryptic peptide derived from digestion of the substrate
protein. The presence of the GG on the side chain of that lysine
prevents cleavage by trypsin at that site, resulting in an internal
modified lysine residue in a formerly ubiquitinated peptide. The
K-ε-GG group is recognized and enriched by using an antibody
specific to K-ε-GG (Fig. 1). It should be noted that modifica-
tion by the ubiquitin-like proteins Nedd8 and ISG15 also results
Namrata D Udeshi, Philipp Mertins, Tanya Svinkina & Steven A Carr
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Correspondence should be addressed to S.A.C. (firstname.lastname@example.org) or
Published online 19 September 2013; doi:10.1038/nprot.2013.120
ubiquitination is essential for the regulation of cellular protein homeostasis. It also has a central role in numerous signaling
events. recent advances in the production and availability of antibodies that recognize the lys--Gly-Gly (K--GG) remnant
produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich
and detect endogenous ubiquitination sites by mass spectrometry (Ms). the following protocol describes the steps required to
complete a large-scale ubiquitin experiment for the detection of tens of thousands of distinct ubiquitination sites from cell lines
or tissue samples. specifically, we present detailed, step-by-step instructions for sample preparation, off-line fractionation by
reversed-phase chromatography at pH 10, immobilization of an antibody specific to K--GG to beads by chemical cross-linking,
enrichment of ubiquitinated peptides using these antibodies and proteomic analysis of enriched samples by lc–tandem Ms (Ms/Ms).
relative quantification can be achieved by performing stable isotope labeling by amino acids in cell culture (sIlac) labeling of
cells. after cell or tissue samples have been prepared for lysis, the described protocol can be completed in ~5 d.
Sample enriched with
Reagent as supplied DMP
Figure 1 | Enrichment of K-ε-GG peptides using anti–K-ε-GG antibody.
After the antibody has been chemically cross-linked to a protein A bead
using DMP, peptides are individually enriched for K-ε-GG peptides using the
anti–K-ε-GG antibody, which recognizes the di-glycyl remnant remaining on
modified lysine residues after trypsin digestion.
© 2013 Nature America, Inc. All rights reserved.
nature protocols | VOL.8 NO.10 | 2013 | 1951
in a GG remnant being retained on modified lysine residues,
which makes ubiquitination, Nedd8ylation and ISG15ylation
indistinguishable on the basis of the tryptic remnant. However,
Kim et al.7 have completed experiments in HCT116 cells show-
ing that >94% of K-ε-GG sites are a result of ubiquitination as
opposed to Nedd8ylation or ISG15ylation. The K-ε-GG enrich-
ment method can be performed with SILAC-labeled samples to
enable relative quantification of protein ubiquitination across
differentially perturbed states6,9.
The K-ε-GG–specific antibody has been used in a growing
number of large-scale experiments, including those that studied
the effects of proteasome or deubiquitinase inhibition on the
ubiquitin landscape5–7,10, that globally identified putative cullin-
RING ligase substrates11 and that specifically identified ubiquiti-
nome alterations dependent on the ubiquitin ligase PARKIN12. In
addition, a recent tissue-specific analysis of ubiquitination sites in
mouse tissues revealed both regulation of core signaling pathways
and tissue-specific networks by ubiquitination13. The K-ε-GG–
specific antibody enables the analysis of protein ubiquitination in
a site-specific manner, which has the potential to reveal the degree
of site specificity or site promiscuity of E3 ligases for substrate
Our previous work presented a refined workflow for routine
detection of tens of thousands of distinct ubiquitination sites
from single samples through methodological improvements made
to both the off-line sample fractionation step as well as the K-ε-
GG enrichment step6,14 (Figs. 1 and 2). Specifically, we showed
that fractionation of samples by basic pH reversed-phase (bRP)
chromatography before the enrichment of K-ε-GG peptides
significantly increases the number of identified and quantified
K-ε-GG sites in SILAC-labeled samples6,9,14 (Fig. 2). Our previous
work also demonstrated that chemical cross-linking of the K-ε-
GG–specific antibody to a solid support considerably reduces the
contamination of antibody fragments and non–K-ε-GG peptides
present in the final enriched samples (Figs. 1 and 3)6. The present
protocol provides complete step-by-step instructions for imple-
menting the procedural workflow for large-scale enrichment and
identification of ubiquitinated peptides using MS.
Urea lysis of cells or tissue
LysC and trypsin
digestion of proteins
bRP fractionation of peptides
1 111098765432 12
Fractions are recombined by a
noncontiguous pooling scheme
Figure 2 | Workflow for preparing samples for K-ε-GG enrichment. Samples
are lysed in urea buffer, digested with LysC and trypsin and fractionated
off-line using bRP chromatography. Fractions are pooled to eight final
fractions for enrichment of K-ε-GG peptides using the K-ε-GG–specific
antibody. Fxn, fraction.
Wash 3× after
Figure 3 | Example of an SDS-PAGE gel used to evaluate the efficiency of
antibody cross-linking to protein A beads. TFA eluates from ~30 µg of
pre– and post–cross-linked anti–K-ε-GG antibody were analyzed by
SDS-PAGE. After cross-linking, more than a tenfold decrease in staining
density for heavy and light chains of the antibody is required for optimal
results. Reproduced from Udeshi et al.6.
Cells or tissue samples (see Reagent Setup for details)
SILAC amino acids (Cambridge Isotope Laboratories)
Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)
Sodium chloride (NaCl)
EDTA (Sigma-Aldrich, cat. no. E7889)
Aprotinin (Sigma-Aldrich, cat. no. A610)•
Leupeptin (Roche, cat. no. 11017101001)
PMSF (Sigma-Aldrich, cat. no. 93482)
2,6 diaminopyradine-3,5-bis(thiocynate) (PR-619; Sigma-Aldrich,
cat. no. SML0430)
Chloroacetamide (CAM; Sigma-Aldrich, cat. no. C0267)
Bicinchoninic acid (BCA) protein assay kit (Pierce, cat. no. 23225)
DTT (Pierce, cat. no. 20291)
Iodoacetamide (IAM; Sigma-Aldrich, cat. no. A3221)
© 2013 Nature America, Inc. All rights reserved.
1960 | VOL.8 NO.10 | 2013 | nature protocols
acKnowleDGMents We thank L. Gaffney for help with illustrations. This work
was supported in part by the Broad Institute of MIT and Harvard and by grants
from the US National Cancer Institute (U24CA160034, part of the Clinical
Proteomics Tumor Analysis Consortium initiative; to S.A.C.) and the National
Heart, Lung and Blood Institute (HHSN268201000033C and R01HL096738;
autHor contrIButIons N.D.U., P.M., T.S. and S.A.C. developed the protocol.
N.D.U. and S.A.C. wrote the manuscript with input from all authors.
coMpetInG FInancIal Interests
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.
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deleterious effects incurred from the presence of antibody fragments and non–K-ε-GG peptides in enriched samples.
A representative SDS-PAGE gel used to assess the cross-linking reaction is shown in Figure 3 (ref. 6). After cross-linking,
the amount of antibody eluted from protein A beads is reduced by >10×.
We have also found that the reduction in sample complexity by off-line bRP fractionation before K-ε-GG enrichment
(Fig. 2) is crucial for increasing the yield of K-ε-GG peptides6. A benefit of using the noncontiguous pooling scheme shown
in Figure 2 is that each pooled bRP fraction has a uniform distribution of hydrophilic and hydrophobic peptides and
therefore will contain similar numbers of K-ε-GG peptides, thereby taking maximum advantage of the available instrument
duty cycle for sequencing ubiquitinated peptides (Fig. 4a)6,14,22,23. A previous analysis of ubiquitination sites, completed in
full biological triplicate, from SILAC-labeled Jurkat cells treated with either MG-132, PR-619 or left untreated showed that
~3,100 distinct K-ε-GG peptides are quantified in each bRP fraction and that non–K-ε-GG peptides make up <50% of the
total number of quantified peptides in each fraction (Fig. 4a)6. We also observed that >75% of peptides typically separate
into only one of the eight bRP fractions and therefore peptide co-elution interference is markedly reduced in the fractionated
samples compared with an unfractionated sample (Fig. 4b). Taken together, the ubiquitin workflow enabled the quantifica-
tion and identification of ~20,000 distinct K-ε-GG sites from a single triple-encoded SILAC-labeled sample (Fig. 4c)6.
Figure 5 shows an example annotated MS/MS spectrum of a K-ε-GG–modified peptide6.