Analytical Chemistry
Article
Our target deconvolution studies comparing structurally
matched iodo and alkyne probes revealed that the protein
targets and sites of labeling of electrophilic probes are readily
identified by our Suzuki−Miyaura platform. Strikingly, nearly
all proteins and cysteines identified by the alkyne-probe 11
were also identified by the iodo probe 10. Interestingly, for in-
cell labeling studies, we found that iodo probe 10 also labeled a
significant number of proteins and peptides not captured by
alkyne probe 11. We ascribe this increased labeling to the
greater lipophilicity of the iodo probe, which likely leads to
increased in-cell probe accumulation and possibly altered
subcellular localization. Of note, our in vitro gel-based ABPP
studies showed comparable labeling profiles and near-identical
banding patterns, indicating that the increased labeling
observed for the iodo probes is likely limited to cell-based
studies.
Uniquely enabled by the observed orthogonality of Suzuki−
Miyaura cross-coupling and CuAAC, we then applied our
findings to develop an innovative multiplexed chemoproteomic
platform (mCSCP) capable of assaying the ligandability of
both cysteine and lysine residues in single experiments. We
first showed that these reactions can function orthogonally
using gel-based ABPP and then extended our findings to mass
spectrometry-based chemoproteomics. Application of our
multiplexed profiling method to the analysis of bifunctional
chemical crosslinkers revealed that the ligandability of both
lysine and cysteine residues can be assayed in a single
experiment, including for dual amino acid-reactive probes,
enabling identification of lysines preferentially labeled by
compounds that also feature a cysteine-reactive warhead.
Decreased coverage of labeled peptides due to the increased
sample complexity caused by multiplexing is one potential
limitation of our approach that could, in part, be addressed
with isobaric labeling reagents.
biological activity is precluded by bulkier substitutions. The
development of more specialized ligands that reduce require-
ments for excess palladium and boronic acid reagents and that
are compatible with Suzuki−Miyaura cross-coupling of aryl
bromide and chloride probes in cell lysates will further increase
the general utility of this chemistry. CuAAC is the gold
standard bio-orthogonal reaction, which has been widely
adopted by diverse communities and applications. The fact
that Suzuki−Miyaura cross-coupling performs comparably to
CuAAC is a testament to the robustness of the reaction. We do
not anticipate that cross-coupling will supplant CuAAC and
instead expect, as shown here, that bio-orthogonal cross-
coupling reactions, including Suzuki−Miyaura, as well as
Sonagashira,75 oxidative Heck,76 and likely additional reac-
tions, can complement CuAAC labeling and add to the bio-
orthogonal toolbox of reactions for ABPP and chemo-
proteomics.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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sı
Sensitivity of model Suzuki−Miyaura cross-coupling
reaction, LC-MS analysis results, structures of probes
and reagents, general workflow for Suzuki−Miyaura
biotinylation, dose-dependent proteome labeling, gel-
based ABPP, time- and temperature-dependent labeling,
concentration-dependent labeling, Suzuki−Miyaura la-
beling, assessment of precatalyst compatibility with PBS
and aryl halogens, and experimental procedures (PDF)
Cellular labeling, peptide records from the dual method
labeling experiment with and without sp3 clean up, dual
probe labeling experiment results, and BSA labeling
experiment results (XLSX)
Our sulfonyl fluoride-substituted compounds represent a
useful advance for the field of crosslinking mass spectrometry.
While sulfonyl fluorides have been used in crosslinking mass
spectrometry experiments previously,72 to our knowledge, this
chemotype has not yet been incorporated into cysteine-
reactive crosslinkers. As sulfonyl fluorides are known to also
react with tyrosine residues, future studies should integrate
tyrosine-reactive probes13 into such multiplexed chemo-
proteomic workflows. While competive chemoproteomic
studies are well-suited to identify high-affinity crosslinking
sites, our findings indicate that such studies may miss lower
efficient crosslinking events, as shown by our crosslinking MS
data using the protein IMPDH2. Given the widespread interest
in chemical probes that function as covalent molecular glues,
our data point to possible challenges associated with
generating compounds that react intermolecularly with high
efficiency simultaneously at two amino acids.
Looking to the future, we can envision a wide range of
additional applications for this chemistry. Multiplexed gel-
based and chemoproteomic studies, such as those described
here, should prove useful for cases where samples are limited,
such as patient-derived samples or rare cell types. Suzuki−
Miyaura cross-coupling chemistry should also extend to the
labeling and enrichment of additional classes of biomolecules,
such as RNA, that are known to be degraded by the copper
employed in click chemistry.73,74 As halogens are ubiquitous in
bioactive molecules, clinical candidates, and drugs, we foresee
the widespread utility of Suzuki−Miyaura cross-coupling
chemistry in target deconvolution studies, particularly when
Competitive m-CSCP data, cysteine- and lysine-
containing peptides labeled by two or more compounds
with an MS1 ratio >3, and lysine-containing peptides
labeled only by bifunctional compounds 15−18, 21, or
22 with an MS1 ratio >3 (XLSX)
AUTHOR INFORMATION
Corresponding Author
■
Keriann M. Backus − Department of Biological Chemistry,
David Geffen School of Medicine and Department of
Chemistry and Biochemistry, UCLA, Los Angeles, California
90095, United States; Molecular Biology Institute, DOE
Institute for Genomics and Proteomics, and Eli and Edythe
Broad Center of Regenerative Medicine and Stem Cell
Research, UCLA, Los Angeles, California 90095, United
States; Jonsson Comprehensive Cancer Center, UCLA, Los
Angeles, California 90095, United States; orcid.org/
Authors
Jian Cao − Department of Biological Chemistry, David Geffen
School of Medicine, UCLA, Los Angeles, California 90095,
United States
Lisa M. Boatner − Department of Biological Chemistry, David
Geffen School of Medicine and Department of Chemistry and
Biochemistry, UCLA, Los Angeles, California 90095, United
2616
Anal. Chem. 2021, 93, 2610−2618