A Chemical Strategy for Protease Substrate Profiling

Article abstract of DOI:10.1016/j.chembiol.2019.03.007

The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position. CHOPS can, in theory, discover substrates for any protease, but is particularly well suited to discover canonical DPP substrates, as cleaved but not intact DPP substrates can be identified by gel electrophoresis or mass spectrometry. Using CHOPS, we show that DPP8 and DPP9, enzymes that control the Nlrp1 inflammasome through an unknown mechanism, do not directly cleave Nlrp1. We further show that DPP9 robustly cleaves short peptides but not full-length proteins. More generally, this work delineates a practical technology for identifying protease substrates, which we anticipate will complement available “N-terminomic” approaches. Proteases regulate countless (patho)physiological processes, but the identification of protease substrates is challenging. Here, Griswold et al. introduce a simple chemoproteomic strategy, termed CHOPS, for profiling protease substrates. Using CHOPS, the authors identify the cleavage specificities of proteases in cellular lysates and show that DPP9 preferentially processes short peptides.

Full text of DOI:10.1016/j.chembiol.2019.03.007

Brief Communication
A Chemical Strategy for Protease Substrate Profiling
Graphical Abstract
Authors
Andrew R. Griswold, Paolo Cifani,
Sahana D. Rao, ...,
Ronald C. Hendrickson, Alex Kentsis,
Daniel A. Bachovchin
Correspondence
bachovcd@mskcc.org
In Brief
Proteases regulate countless (patho)
physiological processes, but the
identification of protease substrates is
challenging. Here, Griswold et al.
introduce a simple chemoproteomic
strategy, termed CHOPS, for profiling
protease substrates. Using CHOPS, the
authors identify the cleavage specificities
of proteases in cellular lysates and show
that DPP9 preferentially processes short
peptides.
Highlights
d
d
d
d
A 2PCA-biotin probe enables chemical enrichment of
protease substrates (CHOPS)
CHOPS rapidly identifies DPP substrates with a gel-based
readout
DPP9 preferentially cleaves short peptides, not whole
proteins (e.g., Nlrp1b)
CHOPS enables unbiased protease substrate profiling with
quantitative proteomics
Griswold et al., 2019, Cell Chemical Biology 26, 1–7
June 20, 2019 ª 2019 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2019.03.007

Please cite this article in press as: Griswold et al., A Chemical Strategy for Protease Substrate Profiling, Cell Chemical Biology (2019), https://doi.org/
0.1016/j.chembiol.2019.03.007
1
Cell Chemical Biology
Brief Communication
A Chemical Strategy
for Protease Substrate Profiling
1
,2
3
4
5
6
6
Andrew R. Griswold, Paolo Cifani, Sahana D. Rao, Abram J. Axelrod, Matthew M. Miele, Ronald C. Hendrickson,
2,3,4,7
2,4,5,8,
Alex Kentsis,
1
and Daniel A. Bachovchin
*
Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, New York, NY 10065, USA
2Pharmacology Program, Weill Cornell Graduate School of Medical Sciences, Memorial Sloan Kettering Cancer Center, New York,
NY 10065, USA
3Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
4Tri-Institutional PhD Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
5Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
6Proteomics Core Laboratory, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
7Department of Pediatrics, Memorial Sloan Kettering Cancer Center and Weill Medical College of Cornell University, New York,
NY 10065, USA
8Lead Contact
*Correspondence: bachovcd@mskcc.org
https://doi.org/10.1016/j.chembiol.2019.03.007
SUMMARY
ond position of polypeptide substrates. DPP4, the best charac-
terized DASH enzyme, cleaves and regulates the activity of
The dipeptidyl peptidases (DPPs) regulate hor- dozens of biologically important peptides, including neuropep-
mones, cytokines, and neuropeptides by cleaving tides, chemokines, and incretins (Mulvihill and Drucker, 2014),
and DPP4 inhibitors are approved anti-diabetic drugs (Deacon
and Lebovitz, 2016). However, many critical substrates of
dipeptides after proline from their amino termini.
Due to technical challenges, many DPP substrates
remain unknown. Here, we introduce a simple
method, termed CHOPS (chemical enrichment of
protease substrates), for the discovery of protease
substrates. CHOPS exploits a 2-pyridinecarboxalde-
hyde (2PCA)-biotin probe, which selectively bio-
tinylates protein N-termini except those with proline
DASH enzymes, including substrates of DPP4, are unknown
Mulvihill and Drucker, 2014; Tagore et al., 2009; Waumans
(
et al., 2015). For example, DPP8 and DPP9 act as an intracellular
checkpoint to restrain the Nlrp1 inflammasome (Okondo et al.,
2017, 2018), but the key substrate that controls inflammasome
activation has not been identified.
DPPs remain poorly characterized in large part due to tech-
in the second position. CHOPS can, in theory, nical challenges in identifying endogenous substrates (Mulvihill
discover substrates for any protease, but is particu- and Drucker, 2014; Tagore et al., 2009; Tinoco et al., 2010;
larly well suited to discover canonical DPP sub- Wilson et al., 2016; Yates et al., 2007). Intact and cleaved
DPP substrates are similar in size and typically inseparable by
strates, as cleaved but not intact DPP substrates
can be identified by gel electrophoresis or mass
spectrometry. Using CHOPS, we show that DPP8
and DPP9, enzymes that control the Nlrp1 inflamma-
some through an unknown mechanism, do not
directly cleave Nlrp1. We further show that DPP9
robustly cleaves short peptides but not full-length
proteins. More generally, this work delineates a
gel electrophoresis, and thus gel-based platforms that exploit
size differences cannot be used for DPP characterization (Dix
et al., 2008; Shao et al., 2007). Moreover, DPPs recognize the
free N-terminal amines of their substrates (Green et al., 2004;
Rasmussen et al., 2003; Ross et al., 2018), limiting the utility
of approaches that involve N-terminal substrate modification
before protease digestion (Tonge et al., 2001; Zhang et al.,
2015). Mass spectrometry (MS)-based global peptide profiling
practical technology for identifying protease sub- (Jost et al., 2009; Tagore et al., 2009; Tammen et al., 2008;
strates, which we anticipate will complement avail- Tinoco et al., 2010, 2011; Yates et al., 2007) and N-terminomics
able ‘‘N-terminomic’’ approaches.
(Kleifeld et al., 2010; Wilson et al., 2013) methodologies have
been used to measure changes in intact and/or cleaved
peptides in response to DPP modulation. However, these
methods do not enrich substrates, and, as such, low abundance
peptides are often difficult to detect (Tagore et al., 2009; Yates
INTRODUCTION
The DPP4 activity and/or structure homolog (DASH) sub-family et al., 2007).
of serine proteases, which include DPP4, DPP7, DPP8, DPP9,
The Francis group recently published an amine-reactive
and FAP, have attracted significant attention as potential thera- chemical group, 2-pyridinecarboxyaldehyde (2PCA, Figure 1A),
peutic targets (Adams et al., 2004; Busek et al., 2004; Lankas which we hypothesized could be used to label and enrich DPP
et al., 2005; Rosenblum and Kozarich, 2003). DASH enzymes substrates from complex lysates (MacDonald et al., 2015).
share the rare ability to cleave after proline residues in the sec- 2PCA condenses with N-terminal amino groups to form imines,
Cell Chemical Biology 26, 1–7, June 20, 2019 ª 2019 Elsevier Ltd.
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Figure 1. A Chemical Strategy for DPP Sub-
strate Profiling
A
(
A) The structure of 2PCA (left), and the biotin-coupled
PCA probe 1 (right).
B and C) 1 forms stable adducts with N-termini (B)
unless proline is in position 2 (C). R can be any side
chain, R can be any side chain except proline. The
gray circles represent the remaining peptide chain.
D) Schematic of the expected results for a DASH and
a non-DASH substrate containing a proline in position
incubated with or without enzyme and labeled
with 1.
E) Full-length PYY(1-36) was incubated with the
indicated enzyme (10 nM) for 4 h before addition of 1
10 mM, 16 h). Cleaved PYY(3-36) was labeled with 1
as a control. See also Figure S1.
F) PYY, NPY, and CXCL12 (10 mM) were incubated
2
(
1
2
(
B
C
2
(
(
(
with PBS, DPP4, 8, or 9 (10 nM) for the indicated time,
before aliquots were removed, boiled, and labeled
with 1 (10 mM). Blots depict probe labeling (strepta-
vidin IR dye) and total protein (Coom.).
D
E
tion for a technology to unbiasedly identify
protease substrates in whole proteomes.
RESULTS
Visualization of DPP Proteolysis by
SDS-PAGE
F
We initially tested whether 1 could detect
the processing of DPP4 substrate peptide
YY (PYY), a 36 amino acid peptide that reg-
ulates postprandial satiety. PYY exists in
two forms, PYY(1-36) and PYY(3-36) (Bat-
terham et al., 2003); the latter is generated
by DPP4-mediated removal of its N-terminal
2
NH -Tyr-Pro dipeptide. We observed that 1
biotinylated PYY(3-36), but not PYY(1-36),
by SDS-PAGE (Figure 1E) and MALDI-TOF
(
Figure S1). Moreover, upon incubating
PYY(1-36) with DPP4, we observed an in-
crease in biotinylation (Figures 1E and S1).
which then cyclize with the next nitrogen in the peptide chain to Notably, the two forms of PYY are indistinguishable by electro-
form stable 5-membered rings (Figure 1B). All N-termini are sta- phoretic migration, but 1 enables PYY cleavage to be visualized
bly labeled, except for those with penultimate amino-terminal by SDS-PAGE without the need for MS.
prolines, which are unable to cyclize (Figure 1C). Importantly,
To further explore the capability of CHOPS to profile DPP sub-
lysine amino groups are not stably modified by 2PCA as they strates in vitro, we examined PYY(1-36) processing by DPP4,
lack nearby amide groups for cyclization. Here, we leverage DPP8, and DPP9. After 1 h, DPP4 treatment resulted in strong la-
this unique reactivity of 2PCA to label cleaved, but not intact beling (Figure 1F), consistent with published kinetic data (Bjelke
DPP substrates (Figure 1D). We show that a biotin-coupled et al., 2006). DPP8 and DPP9 cleave PYY(1-36) much less effi-
2
PCA-based probe (1, Figure 1A) can readily identify cleaved ciently than DPP4 (Bjelke et al., 2006), and accordingly only a
DPP substrates in vitro and selectively enrich cleaved DPP sub- slight increase in biotinylation was observed even after 24 h in
strates from cell lysates. Using this platform, termed chemical DPP8- and DPP9-treated samples (Figure 1F). We next asked
enrichment of protease substrates (CHOPS), we show that whether other DPP substrates were compatible with this
DPP8 and DPP9, enzymes that restrain the Nlrp1 inflammasome, method. Neuropeptide Y (NPY) is critical for nervous system
do not directly cleave Nlrp1b. We furthermore demonstrate that function (Reichmann and Holzer, 2016), and the two N-terminal
2
DPP9 has little activity against full-length proteins but robustly amino acids, NH -Tyr-Pro, are known to be efficiently cleaved
cleaves short peptides. Although CHOPS is particularly useful by DPP4, DPP8, and DPP9 (Bjelke et al., 2006). Indeed, we
for DPP profiling, we show that it is, in theory, compatible with observed that all three enzymes rapidly induced biotinylation of
any protease of interest, and therefore this work lays the founda- NPY (Figure 1F). Chemokine ligand 12 (CXCL12) is a known,
2
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Figure 2. Characterization of Probe 1 Reac-
tivity in Lysates
A
C
D
(
A) Graphical depiction of ubiquitin fusion strategy.
deubiquitinase (DUB), either endogenous in
A
HEK293T cells or recombinantly added to bacterial
lysates, generates the desired N-terminal residue.
(
B and C) 1 labeling (10 mM, 16 h) of purified SMAC
proteins (25 mM) alone (B) or treated with the indi-
cated DPP (10 nM, 16 h) (C).
(
D) Lysates from HEK293T cells expressing the
indicated SMAC proteins were harvested, labeled
with 1 (10 mM, 16 h), and enriched on streptavidin
agarose beads. Immunoblots depict probe labeling
B
(
streptavidin IR dye) and total SMAC protein.
Nlrp1, and the authors of this study simi-
larly speculated, but did not directly
assess, whether DPP9 cleaves the NH
Ser-Pro dipeptide (Zhong et al., 2018). To
albeit slowly cleaved, substrate of DPP4 and DPP8 (Ajami et al., determine if Nlrp1 is a substrate of DPP8/9, we overexpressed
008). Our SDS-PAGE assay confirmed the cleavage kinetics for two C-terminal fragments of mouse Nlrp1b (starting at S984 or
2
-
2
DPP4 and DPP8, and further showed that DPP9, which had not M986), treated lysates from these cells with 1, and enriched bio-
been previously tested for its ability to cleave CXCL12, also tinylated proteins. As expected, only M986-Nlrp1b, which does
slowly cleaves this substrate.
not have a proline in the second position, was enriched (Fig-
ure 3B). We then treated lysates containing S984-Nlrp1b with
DPP8 or DPP9, before performing a similar pull-down experi-
Detection of DPP Substrates in Complex Lysates
We next wanted to determine if CHOPS could identify loss of an ment with 1 (Figure 3C). We did not observe an increase in
N-terminal NH -Xaa-Pro dipeptide from a protein in a cellular Nlrp1b, demonstrating that DPP8/9 do not efficiently remove
lysate. We initially chose to evaluate the pro-apoptotic protein the N-terminal dipeptide.
2
SMAC (Du et al., 2000; Wu et al., 2000), which is proteolytically
It remained possible that DPP8/9 could only cleave this pep-
processed in the mitochondria to reveal an N-terminal IAP-bind- tide bond in the context of the full-length protein. To evaluate if
ing motif (IBM), NH
SMAC proteins starting with V57 (V57-SMAC) or I59 (I59- pressing full-length Nlrp1b. Importantly, these cells were treated
SMAC), which differ only by an N-terminal NH -Val-Pro dipep- with Val-boroPro, a potent inhibitor of DPP4, DPP8, and DPP9
2
-Ala-Val-Pro-Ile, at residue 56. We purified this was the case, we harvested lysates from HEK293T cells ex-
2
tide, using a ubiquitin fusion strategy (Figure 2A) (Bachmair (Okondo et al., 2017), to ensure that any Nlrp1b cleavage by
et al., 1986). As expected, 1 strongly biotinylated purified endogenous DPP8/9, if possible, was blocked (Figure S2). We
I59-SMAC but not V57-SMAC (Figure 2B). DPP4, DPP8, and then treated these lysates with recombinant DPP8 or DPP9,
DPP9 did not increase probe labeling of V57-SMAC (Figure 2C), boiled the samples to separate the N- and C-terminal fragments
indicating that V57-SMAC is not a DPP substrate. Next, we ex- of Nlrp1b, and performed a pull-down experiment with 1. We
pressed both SMAC variants in HEK293T cells, labeled the re- observed no increase in the amount of captured C-terminal frag-
sulting lysates with 1, and enriched the probe-labeled proteins ment (Figure 3D), further indicating that DPP8/9 do not cleave
using streptavidin-coupled beads. Consistent with the purified Nlrp1b. Consistent with this result, CARD8, a homolog of Nlrp1
protein results, only I59-SMAC was enriched (Figure 2D). Impor- that mediates DPP8/9 inhibitor-induced pyroptosis in human
2
tantly, these results demonstrate that this strategy is compatible AML cells (Johnson et al., 2018), has an NH -Ser-Leu sequence
with whole cellular proteomes.
following autoproteolysis, and therefore is unlikely to be directly
cleaved by DPP8/9.
DPP8/9 Do Not Cleave Nlrp1b
Inhibitors of DPP8/9 activate the Nlrp1 inflammasome (Okondo An MS-Based Platform for Proteome-wide Evaluation of
et al., 2017, 2018), which in turn activates caspase-1 and in- Protease Substrates
duces a lytic form of cell death called pyroptosis. However, the We next developed an MS-coupled CHOPS assay to enable the
substrates of DPP8/9 that control the Nlrp1 inflammasome unbiased identification of protease substrates across the entire
remain unknown. Intriguingly, Nlrp1 has a ‘‘function-to-find’’ proteome (Figure 4A). In this format, isotopically labeled cells
(
FIIND) domain, which undergoes post-translational autopro- or cell lysates are subjected to differential proteolysis, mixed,
teolysis and generates N- and C-terminal polypeptide fragments labeled with 1, enriched on streptavidin beads, and digested
that remain associated in an auto-inhibited state (D’Osualdo with trypsin. The probe-labeled peptides are then eluted from
et al., 2011; Finger et al., 2012; Frew et al., 2012) (Figure 3A). the beads and identified and quantified by high-resolution tan-
2
The new N-terminus of the C-terminal fragment is NH -Ser- dem mass spectrometry. Importantly, this format, unlike the
Pro, a dipeptide sequence potentially removed by DPP8/9. SDS-PAGE format, should enable the interrogation of all prote-
DPP9 was also recently reported to directly associate with ases, regardless of cleavage specificity (i.e., an MS readout
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Figure 3. DPP8/9 Do Not Cleave the C-Termi-
nal Fragment of Nlrp1b
A
B
(
A) Graphical depiction of mouse Nlrp1b. The
nucleotide-binding (NACHT), leucine-rich repeat
LRR), function-to-find (FIIND), and caspase acti-
(
vation and recruitment (CARD) domains are shown.
The FIIND domain undergoes post-translational
autoproteolysis generating N- and C-terminal poly-
peptide fragments.
(
B and C) 1 labeling (10 mM, 16 h) and enrichment
of HEK293T lysates ectopically expressing the
indicated Nlrp1b fragment (both with C-terminal
V5 tags) alone (B), or treated with the indicated
DPP (10 nM, 24 h) (C). Immunoblots depict probe
labeling (streptavidin IR dye) and total Nlrp1b
C-terminus (V5).
D
C
(
D) HEK293T cells expressing full-length Nlrp1b with
a C-terminal V5 tag were grown in the presence of
Val-boroPro to ensure that any Nlrp1b cleavage by
DPP8/9 cleavage, if possible, was blocked. See
also Figure S2. Lysates were harvested, treated with
the indicated DPP (10 nM, 24 h), labeled with 1
(
10 mM, 16 h), and enriched on streptavidin
agarose beads. The immunoblot depicts total
Nlrp1b protein (V5).
does not require exclusive labeling of just the cleaved substrate). that DPP9 likely has little activity against full-length proteins.
As such, we predicted that this platform could serve as an alter- Consistent with these data, we observed no change in protein
native to the subtiligase-based platform (Mahrus et al., 2008), biotinylation as observed by SDS-PAGE (Figure S3D). Similarly,
which uses an engineered enzyme (subtiligase) to conjugate a previous study using TAILS (terminal amine isotopic labeling
a biotin-containing glycolate ester chemical probe to protein of substrates) found very few peptides corresponding to
N-termini.
DPP8/9 substrates (Wilson et al., 2013). DPP8/9 can efficiently
As an initial validation of this method, we analyzed the endog- cleave short peptides in vitro (Geiss-Friedlander et al., 2009;
enous N-termini of HEK293T cells and THP-1 cells by mixing Justa-Schuch et al., 2016; Wilson et al., 2013; Zhang et al.,
untreated heavy and light SILAC-labeled lysates before enrich- 2015), and we speculated that DPP8/9 might also cleave short
ing N-termini. We identified 1902 1-labeled-N-termini peptides peptides, but not globular proteins, in cells. We therefore trypsi-
with reproducible relative abundance (i.e., similar heavy:light ra- nized DPP8/9 KO THP-1 lysates to generate peptides before
tios) across two replicates (Figures 4B, S3A, and S3B; Tables treating the heavy-labeled proteome with DPP9 and performing
S1 and S2). The frequencies of the inferred P1 residues preced- CHOPS. In this experiment, we observed 80 heavy-enriched
ing these N-termini closely matched those observed in Jurkat N-termini across two replicates (Figures 4G and 4H), 30 of
cells using subtiligase (Mahrus et al., 2008) (Figure 4C). Interest- which followed inferred P1 prolines (Figures 4H–4J). Overall,
0
ingly, we observed higher frequencies of several P1 residues these data show that CHOPS can indeed identify DPP8/9 sub-
(
Figure 4C), including Pro, Glu, Asp, and Leu, which are disfa- strates in complex mixtures and suggest that DPP8/9 likely act
vored by subtiligase (Weeks and Wells, 2018) but efficiently as peptidases to digest small peptide substrates.
labeled by 2PCA. We next treated heavy-labeled lysates with
trypsin before mixing with untreated light lysates and perform- DISCUSSION
ing CHOPS. We observed a dramatic increase in the number
of heavy-enriched N-terminal-labeled peptides (Figure 4B, Here, we have introduced a chemical strategy to detect and/or
3
periments), with nearly all following an inferred P1 Arg or Lys used with a simple gel-based readout to evaluate N-terminal
,210 in two tryptic experiments versus 24 in two untreated ex- enrich protease substrates called CHOPS. CHOPS can be
0
residue (Figures 4E and 4F). As expected, the P1 residues NH
2
-Xaa-Pro loss from peptides or proteins, corresponding to
largely matched the untreated sample. Together, these data canonical DPP cleavage. Using this gel-based method, we
demonstrate that CHOPS can profile protease substrates in showed that DPP8/9 do not cleave several proteins starting
lysates.
2
with NH -Xaa-Pro, including SMAC and Nlrp1b. Using an MS-
We next wanted to identify DPP9 substrates in lysates. Thus, based readout, we extended these findings to show that DPP9
we treated lysates from heavy-labeled DPP8/9 KO THP-1 cells does not readily cleave full-length proteins, but instead robustly
(
Figure S3C) (Okondo et al., 2017) with DPP9 before mixing with cleaves short peptides. Although DPP8/9 have been speculated
untreated, light-labeled DPP8/9 KO THP-1 lysates and perform- to cleave several full-length proteins, including SYK and AK2
ing CHOPS. We identified only nine enriched 1-labeled N-termi- (Justa-Schuch et al., 2016; Wilson et al., 2013), all confirmatory
nal peptides (of 429) (Figures 4G and 4H). Moreover, none of assays have been conducted exclusively on peptides corre-
these nine peptides followed an inferred P1 Pro, suggesting sponding to the N-terminus of the protein rather than the
4
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A
B
C
E
G
D
F
H
I
J
Figure 4. An MS-Based Platform for Proteome-wide Evaluation of Protease Substrates
(
A) Graphical depiction of MS-CHOPS. ‘‘AcN’’ indicates an acetylated N-terminus, which will not be labeled by 1.
B–F) CHOPS identified 1-labeled peptides from untreated heavy- and light-labeled THP-1 lysates (n = 2 biological replicates; replicate 1 = 863 peptides,
(
replicate 2 = 1,003 peptides), trypsin-treated heavy- and light-labeled THP-1 lysates (n = 2 biological replicates; replicate 1 = 2,219 peptides, replicate 2 = 2,048
peptides), and untreated heavy- and light-labeled lysates HEK293T lysates (n = 2 biological replicates; replicate 1 = 194 peptides, replicate 2 = 353 peptides).
0
Isotopic enrichments from one replicate of the indicated experiments are shown (B). The mean frequency of inferred P1 residues and P1 residues of probe
1
-labeled peptides from the untreated experiments (n = 2) at similar heavy:light ratios (gray box in B) were compared with a previous analysis of Jurkat cells using
0
subtiligase (Mahrus et al., 2008) (C). Sequence logo representation of residues P4–P4 for the THP-1 ‘‘N-terminome’’ determined by CHOPS is shown (D).
0
Frequency of inferred P1 residues and P1 residues (E) and sequence logo representation (F) for heavy-enriched probe-1-labeled peptides (blue box in B) from the
trypsin-treated experiment are shown.
(G–J) CHOPS identified 1-labeled peptides from heavy DPP9-treated and light untreated DPP8/9 KO THP-1 lysates (n = 1 biological replicate; 429 peptides) and
heavy DPP9-treated and light untreated tryptic DPP8/9 KO THP-1 proteomes (n = 2 biological replicates; replicate 1 = 2,390 peptides, replicate 2 = 2,198
peptides). All peptides (G) and heavy-enriched peptides (H, blue box in G) from a single replicate of the indicated experiments are shown. Open circles indicate
inferred Xaa-Pro cleavage, filled circles indicate non-P1 Pro sequences (H). Frequency of inferred P1 residues (I) and sequence logo representation (J) or heavy-
enriched probe-1-labeled peptides from the tryptic DPP9-treated experiment.
full-length protein. Although we cannot rule out that CHOPS strates. However, CHOPS does have a clear limitation in that
0
may have failed to identify a key full-length protein substrate of cannot detect substrates containing a P2 Pro. Future work will
DPP8/9, we speculate that DPP8/9 are enzymes primarily be needed to fully characterize the advantages and disadvan-
involved in the cleavage of cellular peptides.
tages of CHOPS as a general N-terminomic strategy. Regard-
More generally, we have shown that CHOPS can be coupled less, the work here delineates a simple strategy for identifying
with quantitative MS to identify substrates for proteases outside protease substrates in complex lysates and encourages its
the DPP family. In this regard, we predict that CHOPS will form further development and application.
the basis of a complementary N-terminomic strategy to subtili-
gase and TAILS. As CHOPS has more general reactivity with SIGNIFICANCE
N-termini than subtiligase (Figure 4C), it may offer compatibility
with a broader substrate repertoire; as CHOPS involves positive The dipeptidyl peptidases (DPPs) are a family of serine
enrichment, unlike TAILS, it may detect lower abundance sub- proteases that cleave substrates after penultimate
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amino-terminal proline residues. The DPPs cleave and regu-
late a wide range of peptide hormones, cytokines, and neu-
ropeptides, and several have attracted significant interest
as therapeutic targets. However, the DPPs remain poorly
characterized due to technical challenges in discovering
their physiologically relevant substrates, which are often in
low abundance and indistinguishable in size by gel electro-
phoresis after cleavage. Here, we show that 2-pyridinecar-
boxaldehyde (2PCA)-based chemical probes can be used
to selectively label and enrich canonical DPP substrates.
Importantly, this chemical-based strategy, called CHOPS,
not only readily identifies DPP substrates in vitro but also en-
ables the enrichment of cleaved DPP substrates from cell ly-
sates. Using this approach, we show that DPP8 and DPP9,
enzymes that control the Nlrp1b inflammasome through an
unknown mechanism, do not cleave Nlrp1b or other full-
length proteins. More generally, these findings define a use-
ful strategy for the rapid evaluation of DPP substrates and
provide the bases for future development of a chemical
reactivity-based protease profiling platform.
AUTHOR CONTRIBUTIONS
A.R.G. and D.A.B. designed and performed experiments, analyzed data, and
wrote the paper. A.R.G. with assistance from A.J.A. synthesized compounds.
P.C., M.M.M., A.K., and R.C.H. assisted with LC-MS/MS experiments and MS
data analysis. S.D.R. assisted with cloning and transfections.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: June 21, 2018
Revised: February 14, 2019
Accepted: March 12, 2019
Published: April 18, 2019
REFERENCES
Adams, S., Miller, G.T., Jesson, M.I., Watanabe, T., Jones, B., and Wallner,
B.P. (2004). PT-100, a small molecule dipeptidyl peptidase inhibitor, has
potent antitumor effects and augments antibody-mediated cytotoxicity via a
novel immune mechanism. Cancer Res. 64, 5471–5480.
Ajami, K., Pitman, M.R., Wilson, C.H., Park, J., Menz, R.I., Starr, A.E., Cox,
J.H., Abbott, C.A., Overall, C.M., and Gorrell, M.D. (2008). Stromal cell-derived
factors 1alpha and 1beta, inflammatory protein-10 and interferon-inducible
T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8. FEBS
Lett. 582, 819–825.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
Bachmair, A., Finley, D., and Varshavsky, A. (1986). In vivo half-life of a protein
is a function of its amino-terminal residue. Science 234, 179–186.
Batterham, R.L., Cohen, M.A., Ellis, S.M., Le Roux, C.W., Withers, D.J., Frost,
G.S., Ghatei, M.A., and Bloom, S.R. (2003). Inhibition of food intake in obese
subjects by peptide YY3-36. N. Engl. J. Med. 349, 941–948.
d KEY RESOURCES TABLE
d CONTACT FOR REAGENTS AND RESOURCES SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
Bjelke, J.R., Christensen, J., Nielsen, P.F., Branner, S., Kanstrup, A.B.,
Wagtmann, N., and Rasmussen, H.B. (2006). Dipeptidyl peptidases
8
and 9: specificity and molecular characterization compared with dipeptidyl
B Biological Methods
peptidase IV. Biochem. J. 396, 391–399.
B Mass Spectrometry-based CHOPS
B LC-MS/MS Method
Busek, P., Malik, R., and Sedo, A. (2004). Dipeptidyl peptidase IV activity
and/or structure homologues (DASH) and their substrates in cancer. Int. J.
Biochem. Cell Biol. 36, 408–421.
B Proteomic Analysis
B FP-Biotin Enrichment of Serine Proteases
B Chemical Synthesis
Chi, H., Liu, C., Yang, H., Zeng, W.F., Wu, L., Zhou, W.J., Wang, R.M., Niu,
X.N., Ding, Y.H., Zhang, Y., et al. (2018). Comprehensive identification of pep-
tides in tandem mass spectra using an efficient open search engine. Nat.
Biotechnol. https://doi.org/10.1038/nbt.4236.
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
Crooks, G.E., Hon, G., Chandonia, J.M., and Brenner, S.E. (2004). WebLogo: a
sequence logo generator. Genome Res. 14, 1188–1190.
SUPPLEMENTAL INFORMATION
D’Osualdo, A., Weichenberger, C.X., Wagner, R.N., Godzik, A., Wooley, J.,
and Reed, J.C. (2011). CARD8 and NLRP1 undergo autoproteolytic process-
ing through a ZU5-like domain. PLoS One 6, e27396.
Supplemental Information can be found online at https://doi.org/10.1016/j.
chembiol.2019.03.007.
Deacon, C.F., and Lebovitz, H.E. (2016). Comparative review of dipeptidyl
peptidase-4 inhibitors and sulphonylureas. Diabetes Obes. Metab. 18,
ACKNOWLEDGMENTS
3
33–347.
This work was supported by the Josie Robertson Foundation (D.A.B. and A.K.),
a Stand Up to Cancer-Innovative Research grant (grant number SU2C-AACR-
IRG11-17 to D.A.B.; Stand Up to Cancer is a program of the Entertainment
Industry Foundation. Research grants are administered by the American Asso-
ciation for Cancer Research, the scientific partner of SU2C), the Pew Chari-
table Trusts (D.A.B. is a Pew-Stewart Scholar in Cancer Research), the Persh-
ing Square Sohn Cancer Research Alliance (D.A.B.), the NIH (R01 AI137168
to D.A.B.; T32 GM007739-Andersen to A.R.G; R21 CA188881 and R01
CA204396 to A.K; and the MSKCC Core Grant P30 CA008748), an Alfred P.
Sloan Foundation Research Fellowship (D.A.B.), Burroughs Wellcome Fund
Dix, M.M., Simon, G.M., and Cravatt, B.F. (2008). Global mapping of the
topography and magnitude of proteolytic events in apoptosis. Cell 134,
679–691.
Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000). Smac, a mitochondrial pro-
tein that promotes cytochrome c-dependent caspase activation by eliminating
IAP inhibition. Cell 102, 33–42.
Finger, J.N., Lich, J.D., Dare, L.C., Cook, M.N., Brown, K.K., Duraiswami, C.,
Bertin, J., and Gough, P.J. (2012). Autolytic proteolysis within the function to
find domain (FIIND) is required for NLRP1 inflammasome activity. J. Biol.
Chem. 287, 25030–25037.
(
(
A.K), Alex’s Lemonade Stand Foundation (A.K.), Gabrielle’s Angel Foundation
D.A.B. and A.K.), and the Damon Runyon Cancer Research Foundation (A.K.
Frew, B.C., Joag, V.R., and Mogridge, J. (2012). Proteolytic processing of
Nlrp1b is required for inflammasome activity. PLoS Pathog. 8, e1002659.
is the Damon Runyon-Richard Lumsden Foundation Clinical Investigator). We
thank E. Weerapana, J. Peeler, C. Wang, Y. Liu, and K. Boehm for helpful com-
ments and discussion.
Geiss-Friedlander, R., Parmentier, N., Moller, U., Urlaub, H., Van den Eynde,
B.J., and Melchior, F. (2009). The cytoplasmic peptidase DPP9 is rate-limiting
6
Cell Chemical Biology 26, 1–7, June 20, 2019

Please cite this article in press as: Griswold et al., A Chemical Strategy for Protease Substrate Profiling, Cell Chemical Biology (2019), https://doi.org/
0.1016/j.chembiol.2019.03.007
1
for degradation of proline-containing peptides. J. Biol. Chem. 284,
7211–27219.
Ross, B., Krapp, S., Augustin, M., Kierfersauer, R., Arciniega, M., Geiss-
Friedlander, R., and Huber, R. (2018). Structures and mechanism of dipeptidyl
peptidases 8 and 9, important players in cellular homeostasis and cancer.
Proc. Natl. Acad. Sci. U S A 115, E1437–E1445.
2
Green, B.D., Mooney, M.H., Gault, V.A., Irwin, N., Bailey, C.J., Harriott, P.,
Greer, B., O’Harte, F.P., and Flatt, P.R. (2004). N-terminal His(7)-modification
of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-sta-
ble analogues with potent antihyperglycaemic activity. J. Endocrinol. 180,
Shao, W., Yeretssian, G., Doiron, K., Hussain, S.N., and Saleh, M. (2007). The
caspase-1 digestome identifies the glycolysis pathway as a target during
infection and septic shock. J. Biol. Chem. 282, 36321–36329.
379–388.
Johnson, D.C., Taabazuing, C.Y., Okondo, M.C., Chui, A.J., Rao, S.D., Brown,
F.C., Reed, C., Peguero, E., de Stanchina, E., Kentsis, A., et al. (2018). DPP8/
DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.
Nat. Med. 24, 1151–1156.
Tagore, D.M., Nolte, W.M., Neveu, J.M., Rangel, R., Guzman-Rojas, L.,
Pasqualini, R., Arap, W., Lane, W.S., and Saghatelian, A. (2009). Peptidase
substrates via global peptide profiling. Nat. Chem. Biol. 5, 23–25.
Tammen, H., Hess, R., Rose, H., Wienen, W., and Jost, M. (2008). Peptidomic
analysis of blood plasma after in vivo treatment with protease inhibitors–a
proof of concept study. Peptides 29, 2188–2195.
Jost, M.M., Lamerz, J., Tammen, H., Menzel, C., De Meester, I., Lambeir, A.M.,
Augustyns, K., Scharpe, S., Zucht, H.D., Rose, H., et al. (2009). In vivo profiling
of DPP4 inhibitors reveals alterations in collagen metabolism and accumula-
tion of an amyloid peptide in rat plasma. Biochem. Pharmacol. 77, 228–237.
Tinoco, A.D., Kim, Y.G., Tagore, D.M., Wiwczar, J., Lane, W.S., Danial, N.N.,
and Saghatelian, A. (2011). A peptidomics strategy to elucidate the proteolytic
pathways that inactivate peptide hormones. Biochemistry 50, 2213–2222.
Justa-Schuch, D., Silva-Garcia, M., Pilla, E., Engelke, M., Kilisch, M., Lenz, C.,
Moller, U., Nakamura, F., Urlaub, H., and Geiss-Friedlander, R. (2016). DPP9 is
a novel component of the N-end rule pathway targeting the tyrosine kinase
Syk. Elife 5, https://doi.org/10.7554/eLife.16370.
Tinoco, A.D., Tagore, D.M., and Saghatelian, A. (2010). Expanding the dipep-
tidyl peptidase 4-regulated peptidome via an optimized peptidomics platform.
J. Am. Chem. Soc. 132, 3819–3830.
Kleifeld, O., Doucet, A., auf dem Keller, U., Prudova, A., Schilling, O., Kainthan,
R.K., Starr, A.E., Foster, L.J., Kizhakkedathu, J.N., and Overall, C.M. (2010).
Isotopic labeling of terminal amines in complex samples identifies protein
N-termini and protease cleavage products. Nat. Biotechnol. 28, 281–288.
Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J.,
Pognan, F., Hawkins, E., Currie, I., and Davison, M. (2001). Validation and
development of fluorescence two-dimensional differential gel electrophoresis
proteomics technology. Proteomics 1, 377–396.
Lankas, G.R., Leiting, B., Roy, R.S., Eiermann, G.J., Beconi, M.G., Biftu, T.,
Chan, C.C., Edmondson, S., Feeney, W.P., He, H., et al. (2005). Dipeptidyl
peptidase IV inhibition for the treatment of type 2 diabetes: potential impor-
Waumans, Y., Baerts, L., Kehoe, K., Lambeir, A.M., and De Meester, I. (2015).
The dipeptidyl peptidase family, prolyl oligopeptidase, and prolyl carboxypep-
tidase in the immune system and inflammatory disease, including atheroscle-
rosis. Front. Immunol. 6, 387.
tance of selectivity over dipeptidyl peptidases
988–2994.
8 and 9. Diabetes 54,
2
Weeks, A.M., and Wells, J.A. (2018). Engineering peptide ligase specificity by
MacDonald, J.I., Munch, H.K., Moore, T., and Francis, M.B. (2015). One-step
site-specific modification of native proteins with 2-pyridinecarboxyaldehydes.
Nat. Chem. Biol. 11, 326–331.
proteomic identification of ligation sites. Nat. Chem. Biol. 14, 50–57.
Wilson, C.H., Indarto, D., Doucet, A., Pogson, L.D., Pitman, M.R., McNicholas,
K., Menz, R.I., Overall, C.M., and Abbott, C.A. (2013). Identifying natural sub-
strates for dipeptidyl peptidases 8 and 9 using terminal amine isotopic labeling
of substrates (TAILS) reveals in vivo roles in cellular homeostasis and energy
metabolism. J. Biol. Chem. 288, 13936–13949.
Mahrus, S., Trinidad, J.C., Barkan, D.T., Sali, A., Burlingame, A.L., and Wells,
J.A. (2008). Global sequencing of proteolytic cleavage sites in apoptosis by
specific labeling of protein N termini. Cell 134, 866–876.
Mulvihill, E.E., and Drucker, D.J. (2014). Pharmacology, physiology, and
Wilson, C.H., Zhang, H.E., Gorrell, M.D., and Abbott, C.A. (2016). Dipeptidyl
peptidase 9 substrates and their discovery: current progress and the applica-
tion of mass spectrometry-based approaches. Biol. Chem. 397, 837–856.
mechanisms of action of dipeptidyl peptidase-4 inhibitors. Endocr. Rev. 35,
992–1019.
Okondo, M.C., Johnson, D.C., Sridharan, R., Go, E.B., Chui, A.J., Wang, M.S.,
Poplawski, S.E., Wu, W., Liu, Y., Lai, J.H., et al. (2017). DPP8 and DPP9 inhi-
bition induces pro-caspase-1-dependent monocyte and macrophage pyrop-
tosis. Nat. Chem. Biol. 13, 46–53.
Wu, G., Chai, J., Suber, T.L., Wu, J.W., Du, C., Wang, X., and Shi, Y. (2000).
Structural basis of IAP recognition by Smac/DIABLO. Nature 408, 1008–1012.
Yang, X., Boehm, J.S., Yang, X., Salehi-Ashtiani, K., Hao, T., Shen, Y.,
Lubonja, R., Thomas, S.R., Alkan, O., Bhimdi, T., et al. (2011). A public
genome-scale lentiviral expression library of human ORFs. Nat. Methods 8,
Okondo, M.C., Rao, S.D., Taabazuing, C.Y., Chui, A.J., Poplawski, S.E.,
Johnson, D.C., and Bachovchin, D.A. (2018). Inhibition of Dpp8/9 activates
the Nlrp1b inflammasome. Cell Chem Biol 25, 262–267.e5.
6
59–661.
Yates, N.A., Deyanova, E.G., Geissler, W., Wiener, M.C., Sachs, J.R., Wong,
K.K., Thornberry, N.A., Roy, R.S., Settlage, R.E., and Hendrickson, R.C.
Perez-Riverol, Y., Csordas, A., Bai, J., Bernal-Llinares, M., Hewapathirana, S.,
Kundu, D.J., Inuganti, A., Griss, J., Mayer, G., Eisenacher, M., et al. (2019). The
PRIDE database and related tools and resources in 2019: improving support
for quantification data. Nucleic Acids Res. 47, D442–D450.
(
2007). Identification of peptidase substrates in human plasma by FTMS based
differential mass spectrometry. Int. J. Mass Spectrom. 259, 174–183.
Zhang, H., Maqsudi, S., Rainczuk, A., Duffield, N., Lawrence, J., Keane, F.M.,
Justa-Schuch, D., Geiss-Friedlander, R., Gorrell, M.D., and Stephens, A.N.
Rasmussen, H.B., Branner, S., Wiberg, F.C., and Wagtmann, N. (2003). Crystal
structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate
analog. Nat. Struct. Biol. 10, 19–25.
(
2015). Identification of novel dipeptidyl peptidase 9 substrates by two-dimen-
sional differential in-gel electrophoresis. FEBS J. 282, 3737–3757.
Reichmann, F., and Holzer, P. (2016). Neuropeptide Y: a stressful review.
Zhong, F.L., Robinson, K., Teo, D.E.T., Tan, K.Y., Lim, C., Harapas, C.R., Yu,
C.H., Xie, W.H., Sobota, R.M., Au, V.B., et al. (2018). Human DPP9 represses
NLRP1 inflammasome and protects against autoinflammatory diseases via
both peptidase activity and FIIND domain binding. J. Biol. Chem. 293,
18864–18878.
Neuropeptides 55, 99–109.
Rosenblum, J.S., and Kozarich, J.W. (2003). Prolyl peptidases: a serine prote-
ase subfamily with high potential for drug discovery. Curr. Opin. Chem. Biol. 7,
496–504.
Cell Chemical Biology 26, 1–7, June 20, 2019
7

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0.1016/j.chembiol.2019.03.007
1
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
Smac/Diablo Mouse monoclonal Ab
V5 Rabbit polyclonal Ab
DPP9 Rabbit polyclonal Ab
DPP8 Rabbit polyclonal Ab
GAPDH Rabbit monoclonal Ab
IRDye 800CW anti-rabbit
IRDye 800CW anti-Mouse
IRDye 680 RD Streptavidin
Chemicals, Peptides, and Recombinant Proteins
Biotin-coupled 2PCA probe, 1
Gly-Pro-AMC
Cell Signaling Tech
Abcam
2954S; RRID: AB_2131196
Ab9116; RRID: AB_307024
Ab42080; RRID: AB_731947
Ab42076; RRID: AB_731944
14C10; RRID: AB_10693448
925-32211; RRID: AB_2651127
925-32210; RRID: AB_2687825
926-68079
Abcam
Abcam
Cell Signaling Tech
LICOR
LICOR
LICOR
This paper
N/A
VWR
100042-646
Val-boroPro
Okondo et al., 2017
N/A
L-lysine:2HCl
Cambridge Isotope Laboratories
ULM-8766-0.05, lot: I-17807
CNLM-291-H-0.05, lot: PR-26193
ULM-8347-0.1, lot: I1-12740A
CNLM-539-0.1, lot: PR-27681
AS-24401
L-lysine:2HCl 13C6, 99%; 15N2, 99%
L-arginine:HCl
Cambridge Isotope Laboratories
Cambridge Isotope Laboratories
L-arginine:HCl 13C6, 99%; 15N4, 99%
human peptide YY(1-36)
human peptide YY(3-36)
human neuropeptide Y
human chemokine CXCL12/SDF-1 alpha
DPP4
Cambridge Isotope Laboratories
Anaspec
Anaspec
Anaspec
Kerafast
R&D
AS-24405
AS-22464
EMW001
9168-SE-010, Lot: DFQM0417091
BML-SE527-0010, Lot: 05311606
BML-SE528-0010, Lot: 07221432
V5113
DPP8
Enzo Life
Enzo Life
Promega
DPP9
sequencing grade trypsin
Deposited Data
MS-CHOPS proteomics data
Experimental Models: Cell Lines
Human HEK 293T
This paper
ProteomeXchange PXD013019
ATCC
CRL-3216
TIB-202
N/A
Human THP-1
ATCC
Human DPP8/9 KO THP-1
Oligonucleotides
Okondo et al., 2017
Primers for cloning, please see Table S3.
Recombinant DNA
This paper
N/A
pLEX_307
Addgene
Addgene Plasmid Cat #41392
Ubiquitin WT
Addgene
Addgene Plasmid Cat#12647
pET DEST42
ThermoFisher
This paper
This paper
This paper
This paper
This paper
This paper
Cat#12276010
pLEX_307_Ubiquitin_V57-SMAC
pLEX_307_Ubiquitin_I59-SMAC
pET-DEST42_V57-SMAC
pET-DEST42_I59-SMAC
pLEX_307_Ubiquitin_S984-Nlrp1b
pLEX_307_Ubiquitin_M986-Nlrp1b
N/A
N/A
N/A
N/A
N/A
N/A
(Continued on next page)
e1 Cell Chemical Biology 26, 1–7.e1–e6, June 20, 2019

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0.1016/j.chembiol.2019.03.007
1
Continued
REAGENT or RESOURCE
Software and Algorithms
GraphPad Prism version 7
pFIND version 3.1.5
WebLogo 3.0
SOURCE
IDENTIFIER
GraphPad Software
Chi et al., 2018
Crooks et al., 2004
Mestrelab Research
Bruker
www.graphpad.com
www.pfind.ict.ac.cn
www.weblogo.threeplusone.com
www.mestrelab.com
www.bruker.com
Mnova version 11.0.4
FlexControl v3.4
FlexAnalysis v3.4
Bruker
www.bruker.com
ChemDraw Professional 16.0
Perkin Elmer
www.perkinelmer.com
CONTACT FOR REAGENTS AND RESOURCES SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Daniel A.
Bachovchin (bachovcd@mskcc.org).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell lines and cell culture information is listed below. HEK 293T (ATCC, fetal) cells were grown in Dulbecco’s modified Eagle’s medium
(
DMEM) with 10% fetal bovine serum (FBS). THP-1 (ATCC, male) and DPP8/9 KO THP-1 ((Okondo et al., 2017), male) cells were
ꢀ
grown in Roswell Park Memorial Institute medium (RPMI) with 10% FBS. All cells were grown at 37 C in a 5% CO
2
incubator. Cell
lines were regularly tested for mycoplasma using the MycoAlertꢀ Mycoplasma Detection Kit (Lonza). For Stable Isotope Labeling
with Amino acids in Cell culture (SILAC), 50 mL of dialyzed FBS (Sigma-Aldrich) was added to 500 mL RPMI 1640 Media for SILAC
(
Thermo) followed by either 50 mg unlabeled L-Lysine:2HCl, 100 mg unlabeled L-Arginine:HCl or 50 mg labeled L-Lysine:2HCl (13C6,
9%; 15N2, 99%), 100 mg labeled L-Arginine:HCl (13C6, 99%; 15N4, 99%). Media was sterile-filtered with a 0.22 mm filter, aliquoted
9
ꢀ
into sterile 50 mL bottles, labeled light/heavy and stored at 4 C protected from light. All SILAC cells underwent at least 20 cell dou-
blings in respective SILAC media (light/heavy) prior to MS-based experiments.
METHOD DETAILS
Biological Methods
Cloning
cDNA encoding the full-length mouse Nlrp1b gene was cloned from RAW 264.7 macrophages. The full-length gene was shuttled into
a pLEX_307 vector (Addgene) with a C-terminal V5 tag using Gateway technology (Thermo Fisher Scientific). Two C-terminal con-
structs of Nlrp1b starting at S984 and M986 were generated by PCR amplification using specific primers, which contained a 5’
sequence overlap with ubiquitin. The ubiquitin (Ub) sequence was PCR amplified (Addgene 12647) with primers containing a 3’ over-
lap with corresponding Nlrp1b C-termini. The partially overlapping Ub and Nlrp1b C-termini products were mixed and assembled by
PCR to yield the ubiquitin fused constructs. These products were then shuttled into a pLEX_307 vector (Addgene) with a C-terminal
V5 tag using Gateway technology (Thermo Fisher Scientific). cDNA encoding SMAC was cloned from The Broad Institute’s ORFeome
(
Yang et al., 2011), and similarly amplified to generate the ubiquitin fused constructs. These products were then shuttled into a modi-
fied pLEX_307 vector (Addgene) with a C-terminal FLAG tag and a pET-DEST42 vector (Addgene) using Gateway technology
Thermo Fisher Scientific).
(
GP-AMC Assay
DPP4, DPP8, and DPP9 were diluted to 100 nM in PBS with 10% glycerol. A solution of substrate (1 mM Gly-Pro-AMC) was prepared
in DMSO. 16 mL of PBS or the indicated cell lysate was added to a 394-well black clear-bottom plate (Corning), followed by 2 mL of
100 nM enzyme and 2 mL of 1 mM substrate. A blank well with 2 mL of 1 mM substrate and 18 mL of PBS was used as a negative
control. Fluorescence was recorded at ambient temperature every minute for 30 min at 380-nm excitation and 460-nm emission
wavelengths.
Gel-Based In Vitro DPP Substrate Assays
Lyophilized peptide YY(1-36), peptide YY(3-36), neuropeptide Y, and Human Chemokine CXCL12 were resuspended in water to
ꢀ
1
1
00 mM and stored at ꢁ20 C. Enzymatic activity was confirmed by GP-AMC assay. Reactions were prepared with 8 mL of
00-mM peptide stock, 8 mL of 100 nM enzyme (DPP4, DPP8 and DPP9) or PBS (for control), and 64 mL of PBS (Corning). Reactions
ꢀ
incubated at 37 C for 24 h. 9 mL aliquots were taken at 0, 1, 4, 8 and 24 h and immediately boiled to deactivate the enzyme. 1 mL of
ꢀ
1
boiled and run on 16% tricine gels (Invitrogen) at 125 V for 90 min. A semi-dry transfer at 25 V, 2.5 A for 15 min was used to transfer
00 mM 1 was added to the aliquots. After 16 h of incubating at 37 C, an equal volume of 23 loading dye was added, samples were
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0.1016/j.chembiol.2019.03.007
1
proteins to the membrane, which was then blocked and blotted for biotin with streptavidin IR dye. Additional 9 mL aliquots were also
taken at 0, 24 h for Coomassie staining (Invitrogen) according to the manufacturer’s specifications.
Detection of DPP Substrates in Lysates
5
HEK 293T cells were seeded at 5 3 10 cells/well in 6-well plates. The next day cells were transfected by manufacturer’s instructions
(
Fugene, Promega) with the ubiquitin-fused construct (2 mg/well) or red fluorescent protein (mock). Cells were harvested 48 h later,
ꢀ
pelleted by centrifugation (400 3 g, 4 C), washed twice with cold PBS (Corning), resuspended in 250 mL PBS, sonicated (3 3 10 s),
and clarified by centrifuging 15 min at 15,000 3 g. The resulting soluble proteins were transferred to a new microcentrifuge tube; the
concentration was measured by DC protein assay kit (Bio-Rad), and diluted to 1.5 mg/mL.
To test probe labeling of these constructs, 5 mL of 100 mM 1 was added to a 50 mL aliquot of 1.5 mg/mL lysate. After incubating for
ꢀ
1
6 h at 37 C unbound probe was removed by buffer exchanging into PBS using a 3-kDa cut-off ultra-centrifugal filter unit (Amicon)
according to the manufacturer’s instructions. The resulting concentrated solution was then boiled in 100 mL of 1% SDS for 10 min and
diluted to 500 mL with the addition of PBS. 100 mL of high capacity neutroavidin agarose resin slurry (Pierce) was added and the
mixture was rotated end-over-end for 2 h at ambient temperature. Agarose resin was pelleted by centrifugation (500 3 g for
1
2
min), washed 3 times with 0.2% SDS in PBS (1 mL), and 3 times with PBS (1 mL). Boiling the resin with an equivalent volume of
3 SDS loading dye for 10 min eluted the immunoprecipitated peptides, which were separated by SDS-PAGE gel, immunoblotted,
and visualized using the Odyssey Imaging System (LI-COR).
For the DPP8/9 cleavage assay of ubiquitin-fusion generated fragments, 15 mL of 100 nM DPP8, DPP9, or PBS (control) were
ꢀ
added to a 135 mL aliquot of 1.5 mg/mL lysate. The mixture incubated 24 h at 37 C. The samples were boiled to deactivate enzymes,
ꢀ
1
5 mL of 100 mM 1 was added and the reaction incubated at 37 C for 16 h. Unbound probe was removed by buffer exchanging into
PBS using a 3-kDa cut-off ultra-centrifugal filter unit (Amicon) according to the manufacturer’s instructions. The samples then pro-
cessed as described above.
5
For evaluating DPP8/9 cleavage of full-length Nlrp1b, HEK 293T cells were seeded at 5 3 10 cells/well in 6-well plates. The next
day, cells were transiently transfected with the indicated constructs encoding full-length Nlrp1b protein. The cells were then cultured
in the presence of Val-boroPro (10 mM, with fresh drug added each day) for 3 days before the soluble proteome was harvested as
described above. These cells were cultured with Val-boroPro to ensure that any potential DPP cleavage activity on the neo N-termi-
nus generated by Nlrp1b autoproteolysis was blocked (i.e., it would not be possible to see an increase in probe 1 labeling after re-
combinant DPP9 treatment if endogenous DPP9 had already cleaved this substrate completely). Treatment with Val-boroPro was
unnecessary for the ubiquitin-fusion generated C-terminal fragment constructs because a control fragment lacking the N-terminal
NH
2
-Ser-Pro dipeptide could be generated for comparison. Inhibition of endogenous DPP activity was confirmed by GP-AMC assay
(Figure S2A). 20 mL of 100 nM DPP8, DPP9, or PBS (control) were then added to a 200 mL aliquot of 1.5 mg/mL lysate, and activity of
ꢀ
recombinant DPP9 in the lysate was confirmed by GP-AMC assay (Figure S2B). The mixtures were incubated 24 h at 37 C before
being boiled for 10 min to separate the N- and C- terminal fragments. 20 mL of 100 mM 1 was added and the reaction incubated
ꢀ
at 37 C for 16 h. Unbound probe was removed by buffer exchanging into PBS using a 3-kDa cut-off ultra-centrifugal filter unit (Ami-
con) according to the manufacturer’s instructions. The samples were then processed as described above.
Mass Spectrometry-based CHOPS
SILAC labeled (light/heavy) cell pellets were suspended in 25 mM ammonium bicarbonate (ABC) and sonicated (3 3 10 s). The
resulting proteomes were transferred to a new microcentrifuge tubes; the concentration was measured by DC protein assay kit
(
Bio-Rad).
To evaluate the native N-terminome 100 mL of heavy and light lysates (HEK 293T 1 mg/mL, THP-1 2 mg/mL) were mixed together
ꢀ
and labeled with 1 (5 mM) for 16 h at 37 C. Proteins were precipitated by adding seven volumes (1.4 ml) of acetone, vortexing briefly,
ꢀ
ꢀ
incubating for 1 h at ꢁ20 C, centrifuging 10 min at 15,000 3 g (4 C) and decanting the supernatant. The residual protein pellet was
suspended with sonication in 1 mL of 50 mM Tris HCl, 150 mM NaCl with 1% NP-40. 200 ml of high capacity neutroavidin agarose
resin slurry (Pierce) was added and the mixture was rotated end-over-end for 2 h at ambient temperature. Agarose resin was pelleted
by centrifugation (500 3 g for 1 min), washed 2 times with 50 mM Tris HCl, 150 mM NaCl with 1% NP-40 (1 ml), 2 times with PBS
ꢀ
(
1 mL), 2 times with water (1 mL). Proteins were reduced on-bead with 10 mM DTT (42 C for 30 min), and alkylated with 20 mM io-
ꢀ
doacetamide (37 C for 30 min). Agarose resin was pelleted, resuspended in 2 M urea, 1 mM CaCl in 25 mM ABC and trypsinized
ꢀ
overnight (2 ug, Pierce) while shaking at 37 C. The resulting tryptic peptides were washed once with 50 mM Tris HCl, 150 mM
NaCl with 1% NP-40 (1 mL), 2 times with PBS (1 mL), and 2 times with water (1 mL). Bound peptides were eluted twice with
2
ꢀ
2
00 mL of 80% acetonitrile in water containing 0.1% formic acid (first at RT for 10 min, second at 70 C for 10 min). The combined
elution fractions were dried using a Genevac EZ-2 evaporator, resuspended in 100 mL water containing 0.1% formic acid, desalted
with a C18 Silica MicroSpin Column (The Nest Group) according to the manufacturer’s instructions and re-dried using a Genevac
EZ-2 evaporator to give the final peptide pellet for LC-MS/MS analysis.
To evaluate tryptic protease substrates, 200 mL of 3 mg/mL SILAC labeled THP-1 proteomes were boiled for 15 min and sonicated
to resuspend precipitated proteins. Sequencing grade trypsin (2 mg) was added along with 1 mM CaCl
ꢀ
2
to the heavy proteome, an
equal volume of PBS was added to the light proteome. The samples shook overnight at 37 C, at which time the samples were boiled
to inactivate trypsin, sonicated and combined pairwise. Samples were labeled with 1 and processed as above.
To evaluate DPP9 whole proteome substrates, 200 mL of 3 mg/mL SILAC labeled DPP8/9 KO THP-1 proteome was incubated with
ꢀ
either PBS (light) or DPP9 (200 U). The samples shook overnight at 37 C, at which time the samples were boiled to inactivate DPP9,
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0.1016/j.chembiol.2019.03.007
1
sonicated and combined pairwise. Samples were labeled with 1 and processed as above. Rather than trypsinizing on bead, labeled
peptides/proteins were eluted with acetonitrile, dried, trypsinized in solution and then desalted for LC-MS/MS analysis.
To evaluate DPP9 tryptic proteome substrates, 200 mL of 3 mg/mL SILAC labeled DPP8/9 KO THP-1 proteome were trypsinized for
ꢀ
8
shook overnight at 37 C, at which time the samples were boiled to inactivate DPP9, sonicated and combined pairwise. Samples were
h at 37 C. Samples were boiled to inactivate trypsin, sonicated and incubated with either PBS (light) or DPP9 (200 U). The samples
ꢀ
labeled with 1 and processed as above.
LC-MS/MS Method
The desalted peptides were resuspended in 50 ul of 0.1% formic acid in water. 1 ul sample was injected for microcapillary liquid chro-
matography with tandem mass spectrometry using the NanoAcquity (Waters) with a 100- mm-inner-diameter 3 10-cm-length C18
column [1.7 (mm) BEH130, Waters] configured with a 180-mm 3 2-cm trap column coupled to a Q Exactive Plus mass spectrometer
(Thermo Fisher Scientific). Trapping was performed at 15 uL/min (0.1% formic acid) for 1 min. Peptides were eluted with a linear
gradient of 0-50% acetonitrile (0.1% formic acid) in water (0.1% formic acid) over 90 min with a flow rate of 300 (nL/min). Full
scan MS1 spectra were acquired over 400-1600 (m/z) at 70,000 resolution with max IT of 50 ms and automatic gain control
6
AGC) at 1 3 10 ions. MS data was collected in data dependent acquisition (DDA) mode scanning the top 10 most intense precursor
(
ions for HCD fragmentation performed at normalized collision energy (NCE) 27% with AGC at 5 3 10 ions, isolation window 1.5 m/z,
and dynamic exclusion of 15s. MS/MS spectra were collected with resolution of 17,500.
4
Proteomic Analysis
MS/MS data (.raw files) were analyzed with pFIND version 3.1.5 (Chi et al., 2018). Probe 1 was added as a variable peptide N-terminal
5 2 27 1
modification (C21N O H S , +413.1885) and searches were conducted with the following parameters: open search against semi-
tryptic peptides, allowing precursor and fragment tolerances of ± 20 ppm, using a human SwissProt FASTA database with reverse
sequence decoys and common contaminants, quantified with SILAC labeling (Light label = none, Heavy label = SILAC-Arg10Lys8),
and allowing an FDR of less than 1% at the peptides level. The median SILAC ratio and spectra score was calculated for unique pep-
2
tide sequences identified with the correct N-terminal modification and at least 2 spectral counts in a single run. Log (median SILAC
ratio) were calculated setting the upper limit to 5 and lower limit to -5. The P1-P6 residues were determined for the resulting peptide
sequences by searching against the human SwissProt FASTA database, given the identified protein in pFIND. Peptides were clas-
sified as ‘heavy enriched’ given a log
2
(median SILAC ratio) of greater than 2 and ‘light enriched’ given a log
2
(median SILAC ratio) of
less than -2. Peptides were considered ‘at similar level’ given a log
generated using WebLogo (Crooks et al., 2004).
2
(median SILAC ratio) between -2 and 2. Sequence logo were
FP-Biotin Enrichment of Serine Proteases
THP-1 cells were pelleted at 400 g, resuspended in 1 mL of PBS and lysed by sonication. Protein concentrations were determined
using the DC Protein Assay kit (Bio-Rad) and adjusted to 2 mg/mL. One milliliter of 1 mg/mL lysate was reacted with the FP-biotin
ꢀ
probe (5 mM) for 1 h at 25 C. Triton-X100 was then added to a final concentration of 1%, and samples were incubated for 1 h while
ꢀ
rotating at 4 C to solubilize membranes. Lysates were then separated from free probe by passage over a Sephadex G-25M column
ꢀ
(GE Healthcare). SDS was added to a final concentration of 0.5% (in 3.5 mL total volume), and samples were boiled for 10 min at 95 C
to denature proteins. Samples were then diluted in PBS to a final volume of 11 mL before the addition of 200 mL of NeutrAvidin-
ꢀ
Agarose resin (Pierce) and incubation for 1 h while rotating at 25 C. Samples were then washed with 4 3 10 mL PBS and eluted
ꢀ
by resuspension in 150 mL 23 sample loading dye and boiling at 95 C for 10 min.
Purification of SMAC Proteins
Rosetta 2 (DE3) cells (Novagen) containing SMAC constructs in pET-DEST42 vectors were grown in TB media containing 75 mg/L
ꢀ
carbenicillin with shaking at 37 C to an OD600 of 0.5. The cells were then induced with 1 mM IPTG and harvested 4 h later by centri-
ꢀ
fugation. Cells were lysed by stirring for 20 min at 4 C in DUB enzyme buffer (50 mM HEPES, pH 7.5, 0.5 mM EDTA, 1 mM DTT) sup-
plemented with 1 mg/mL lysozyme. The lysate was then sonicated and centrifuged at 10,000 3 g for 10 min. 15 mL of USP2 (60 nM
final concentration) was added to the lysates, which were incubated at room temperature for 5 h. Lysates were then buffer exchanged
using Sephadex G-25M columns (GE Healthcare). Talon cobalt affinity resin (Clontech; 400 mL of slurry/g of cell paste) was added to
ꢀ
the supernatant, and the mixture was rotated at 4 C overnight. Beads were collected by centrifugation at 700 3 g for 3 min, washed
three times with PBS, and applied to a 1 cm column. The column was washed twice with PBS buffer (10 mL/400 mL of resin slurry). The
bound protein was eluted by the addition of 200 mM imidazole (2 mL/400 mL of resin). Imidazole was removed by passage over a
Sephadex G-25M column (GE Healthcare). Protein concentrations were determined using the Bio-Rad DC Protein Assay kit. Glycerol
ꢀ
was added to a final concentration of 10%, and proteins were aliquoted stored at ꢁ80 C until use.
MALDI-TOF
DHBA matrix was prepared freshly by saturating 2,5-Dihydroxybenzoic acid (Sigma) in 40% acetonitrile in water with 0.1% TFA.
10 mM peptide samples in water were diluted 1:10 with 5% acetonitrile in water with 0.1% TFA. 0.5 mL of the resulting solution
was spotted on the MALDI plate followed by 0.5 mL of saturated DHBA matrix. MALDI-TOF was conducted on a Bruker Autoflex instu-
ment. Data was collected and analyzed using ‘‘Compass for flexSeries’’ v1.4, ‘‘flexControl’’ v3.4 and ‘‘flexAnalysis’’ v3.4 software.
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1
Chemical Synthesis
All reagents used for chemical synthesis were purchased from Sigma-Aldrich or Alfa Aesar unless otherwise specified and used
without further purification. All anhydrous reactions were performed under nitrogen atmosphere. Analytical thin-layer chromatog-
raphy (TLC) was conducted on EMD Silica Gel 60 F254 plates with detection under UV (254 nm) or by staining with potassium
4
permanganate (KMnO ). NMR spectra were recorded on a Bruker UltraShield Plus 500 MHz Avance III NMR. Chemical shifts are re-
corded in ppm (d) relative to solvent. Coupling constants (J) are reported in Hz. NMR spectra were processed using Mnova (www.
mestrelab.com/software/mnova-nmr) software. High-resolution mass spectra were obtained on a Waters Acuity Premiere XE TOF
LC-MS by electrospray ionization.
Scheme S1. Synthetic route for 5 according to literature procedure (MacDonald et al., 2015)
Compound 3
6
2
-(hydroxymethyl)-2-pyridinecarboxaldehyde (MacDonald et al., 2015). To a stirred solution of 2,6-pyridinedimethanol 2 (3.0 g,
1.6 mmol) in 1,4-dioxane (60 ml) was added selenium dioxide (1.2 g, 10.8 mmol). The mixture was sonicated for 2 min and then
ꢀ
stirred at 65 C for 24 h. The reaction was cooled to ambient temperature, diluted with dichloromethane (100 ml), filtered through
a plug of Celite, and concentrated under reduced pressure. This crude material was then purified by flash chromatography using
1
.5% methanol in dichloromethane to afford 2.63 g (89%) of the title compound as an off-white solid. H NMR (500 MHz, Chloro-
2
form-d): d 10.08 (s, 1H), 7.92–7.85 (m, 2H), 7.57–7.51 (m, 1H), 4.86 (s, 2H), 3.34 (br s, 1H). C NMR (500 MHz, Chloroform-d):
13
+
7 8 1 2
d 193.2, 160.1, 151.7, 137.7, 125.1, 120.6, 64.1. HRMS (ESI) calculated for C H N O ([M+H] ) 138.0550, found 138.0553.
Compound 4
6
-(formylpyridin-2-yl)methyl methanesulfonate (MacDonald et al., 2015). Methanesulfonyl chloride (1.8 ml, 22.8 mmol) was added
ꢀ
dropwise at 0 C to a stirred solution of 6-(hydroxymethyl)-2-pyridinecarboxaldehyde 3 (2.63 g, 19 mmol) and triethylamine
5.4 ml, 57 mmol) in dichloromethane (65 ml). After 1 h, the reaction was quenched with saturated aqueous sodium bicarbonate.
(
The aqueous layer was separated and extracted three times with dichloromethane. The organic layers were combined, dried over
sodium sulfate, filtered and concentrated under reduced pressure to afford 3.25 g (78%) of the title compound as a brown oil.
1
13
H NMR (400 MHz, Chloroform-d): d 10.03 (s, 1H), 7.96–7.92 (m, 2H), 7.75–7.72 (m, 1H), 5.40 (s, 2H), 3.13 (s, 3H). C NMR
+
([M+H] )
(
100 MHz, CDCl
3
): d, 192.6, 154.5, 152.6, 138.6, 126.4, 121.9, 70.6, 38.1. HRMS (ESI) calculated for C
8
H
10
N
1
O
4
S
1
2
16.0325, found 216.0321.
Compound 5
Tert-butyl 4-((6-formylpyridin-2-yl)methyl)piperazine-1-carboxylate (MacDonald et al., 2015). To a stirred solution of 6-(formylpyridin-
2
-yl)methyl methanesulfonate 4 (3.2 g, 15 mmol) and 1-Boc-piperazine (3.25 g, 15 mmol) in acetonitrile (65 ml) was added potassium
ꢀ
carbonate (4.23 g, 30 mmol). The mixture was stirred at 60 C for 16 h at which time the solvent was removed under reduced pressure.
The resulting residue was then partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The aqueous layer
was separated and extracted three times with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered
and concentrated under reduced pressure. The crude material was purified by flash chromatography with 33% ethyl acetate in hex-
1
anes to afford 2.18 g (48%) of the title compound as a tan solid. H NMR (500 MHz, Chloroform-d) v 10.0 (s, 1H), 7.81 – 7.75 (m, 2H),
7
13
.63 (p, J = 4.0 Hz, 1H), 3.71 (s, 2H), 3.40 (t, J = 5.1 Hz, 4H), 2.42 (t, J = 5.1 Hz, 4H), 1.39 (s, 9H). C NMR (500 MHz, Chloroform-d) v
93.59, 159.43, 154.75, 152.38, 137.45, 127.40, 120.31, 79.71, 64.10, 53.12, 43.65, 28.42. HRMS (m/z) calculated for C16
1
23 3 3
H N O
+
M+H ] 306.1812, found 306.1809.
[
Scheme S2. Synthetic route for 1
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1
Compound 1
-((4-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoyl) piperazin-1-yl) methyl)picolinaldehyde. Com-
6
pound 5 (153 mg, 0.5 mmol) was dissolved in dichloromethane (5 ml), to which 4M HCl in 1,4 dioxane (1.4 ml) was added and the
reaction was allowed to stir for 2 h at room temperature. The resulting white precipitate was then concentrated under vacuum
and the solids were triturated with dichloromethane and dried under reduced pressure. The resulting hydrochloride salt was sus-
pended in acetonitrile (4 ml) with biotin-NHS-ester (204 mg, 0.6 mmol). Triethylamine (0.21 ml, 1.5 mmol) was added dropwise
and the solution was left stirring at room temperature overnight. After 16 h, the solvent was removed under reduced pressure and
the resulting residue was partitioned into dichloromethane and saturated aqueous sodium bicarbonate. The aqueous layer was sepa-
rated and extracted three times with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The crude material was purified by flash chromatography on a Teledyne ISCO CombiFlash
with a gradient of methanol (5% to 15%) in dichloromethane to afford 56.3 mg (26%) of the title compound as an off-white solid.
1
H NMR (500 MHz, Chloroform-d) v 10.03 (s, 1H), 7.84 (d, J = 5.7 Hz, 2H), 7.71 – 7.63 (m, 1H), 6.25 (s, 1H), 5.78 (s, 1H), 4.47
dd, J = 7.8, 4.9 Hz, 1H), 4.27 (dd, J = 8.0, 4.5 Hz, 1H), 3.75 (s, 2H), 3.61 (d, J = 5.3 Hz, 2H), 3.48 (t, J = 5.0 Hz, 2H), 3.13
q, J = 2.9 Hz, 1H), 2.92 – 2.80 (m, 1H), 2.70 (d, J = 12.7 Hz, 1H), 2.49 (dt, J = 19.0, 5.1 Hz, 4H), 2.33 (t, J = 7.5 Hz, 2H), 1.73 –
(
(
1
.61 (m, 4H), 1.44 – 1.36 (m, 2H). C NMR (500 MHz, Chloroform-d) v 192.53, 170.50, 162.93, 158.16, 151.38, 136.55, 126.45,
3
1
1
19.45, 62.88, 60.87, 59.19, 54.50, 52.46, 52.01, 44.56, 40.48, 39.55, 31.74, 27.45, 27.30, 24.13. HRMS (m/z) calculated for
+
S [M+H ] 432.2069, found 432.2062.
C
21
H
29
N
5
O
3
QUANTIFICATION AND STATISTICAL ANALYSIS
Proteomic statistical analysis was performed with pFIND. GraphPad Prism 7 software was used for SEM calculations. WebLogo 3.0
was used to generate sequence logos. For in vitro cell assays, n refers to number of replicate wells analyzed for each treatment group.
Error bars for in vitro assay figures represent SEM from mean.
DATA AND SOFTWARE AVAILABILITY
MS-CHOPS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-
Riverol et al., 2019). The accession number for the data reported is: PXD013019.
Cell Chemical Biology 26, 1–7.e1–e6, June 20, 2019 e6