Useful Tools for Biomolecule Isolation, Detection, and Identification: Acylhydrazone-Based Cleavable Linkers

Article abstract of DOI:10.1016/j.chembiol.2009.06.005

Proteomic searches using affinity-based chromatography (e.g., biotin-[strept]avidin) have been severely hampered by low protein recovery yields, protein destruction and denaturation, and the release of background proteins from the support. These limitations confound protein identification. A new acylhydrazone-based cleavable linker was developed to permit the efficient isolation of proteins with a traceable tag allowing detection and identification under mild conditions. The utility of the acylhydrazone linker was validated in a proteomic search wherein aldehyde dehydrogenase-1 was selectively captured and isolated from the mouse soluble liver proteome without interfering background proteins. The use of acylhydrazone linkers is expected to be generalized, allowing for the selective release of tagged molecules from noncovalent and covalently tagged supports.

Full text of DOI:10.1016/j.chembiol.2009.06.005

Chemistry & Biology
Article
Useful Tools for Biomolecule Isolation,
Detection, and Identification:
Acylhydrazone-Based Cleavable Linkers
1,3,
Ki Duk Park,1 Rihe Liu,1,2, and Harold Kohn
*
*
1Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy
2Carolina Center for Genome Sciences
3Department of Chemistry
University of North Carolina, Chapel Hill, NC 27599, USA
*Correspondence: rliu@email.unc.edu (R.L.), hkohn@email.unc.edu (H.K.)
DOI 10.1016/j.chembiol.2009.06.005
SUMMARY
et al., 1990), a situation that makes it difficult to identify low-
abundance proteins.
Proteomic searches using affinity-based chromatog-
raphy (e.g., biotin-[strept]avidin) have been severely
hampered by low protein recovery yields, protein
destruction and denaturation, and the release of
background proteins from the support. These limita-
tions confound protein identification. A new acylhy-
drazone-based cleavable linker was developed to
permit the efficient isolation of proteins with a trace-
able tag allowing detection and identification under
mild conditions. The utility of the acylhydrazone
linker was validated in a proteomic search wherein
Several methods have been advanced to address the difficulty
of the biotinylated product release step. They include using
either biotin analogs (Hirsch et al., 2002; Zeheb and Orr, 1986)
or protein-engineered streptavidin mutants (Howarth et al.,
2006; Malmstadt et al., 2003; Morag et al., 1996; Wu and
Wong, 2005), structural modifications that weaken the biotin-
(strept)avidin interaction. Although these methods improve the
release of biotinylated molecules, the reduced association
constant for the complex does not allow the application of strin-
gent wash conditions to remove nonspecific protein interactions
from the resin and adversely affects the capture efficiency of the
biotin-tagged adducts. Another approach has been to use
aldehyde dehydrogenase-1 was selectively captured conditions that disrupt the biotin-(strept)avidin complex (Holm-
berg et al., 2005; Jenne and Famulok, 1999; Tong and Smith,
1992). Unfortunately, several of these conditions (e.g., 1%
aqueous SDS/heat) are harsh and generally lead to the release
and isolated from the mouse soluble liver proteome
without interfering background proteins. The use of
acylhydrazone linkers is expected to be generalized,
allowing for the selective release of tagged mole-
of abundant proteins that nonspecifically bind to the matrix or
streptavidin. Furthermore, the recovery yields of the biotinylated
cules from noncovalent and covalently tagged
materials are still low and the captured molecules are often either
supports.
destructed or denatured. A third strategy has been to use bioti-
nylated agents that incorporate a cleavable site between the
biotin and the bound (bio)molecule. Examples include linkers
that contain a proteolytic (Dieterich et al., 2007; Speers and Cra-
INTRODUCTION
In recent years, significant progress has been made in the iden- vatt, 2005) or a chemically labile (e.g., disulfide (Finn et al., 1985;
tification of protein-ligand, protein-drug, and protein-protein Marie et al., 1990; Shimkus et al., 1985), acid-sensitive (van der
interactions (Berggard et al., 2007; Walsh and Chang, 2006). Veken et al., 2005), base-sensitive (Ball and Mascagni, 1997;
Nonetheless, the complexity of the proteome, the low abun- Kazmierski and McDermed, 1995), nucleophile-sensitive (Lin
dance of many proteins, and the temporal and spatial distribu- and Morton, 1991), photolytic (Bai et al., 2004; Thiele and Fah-
tion of proteins make protein target identification difficult. renholz, 1994), reductive (Verhelst et al., 2007) site.
However, new methods have been advanced to identify proteins
We looked for an easy-to-prepare, cleavable linker that would,
involved in biochemical and regulatory pathways. Common to under mild conditions, efficiently release the (bio)molecules teth-
many of these is the selective capture of the tagged protein of ered to a support and for which destruction and/or denaturation
interest from the biological milieu using affinity-based chroma- of the (bio)molecules would be minimal. Ideally, the cleavage
tography (Scriba, 2004). Because the biotin-avidin association reaction would allow the incorporation of a traceable tag (i.e.,
is among the tightest noncovalent interactions known (KA
1014ꢀ1015
=
isotopic, radioactive, fluorescent) that permits the detection
M
ꢀ1) (Green, 1990), biotinylated tags and (strept)avi- and identification of the captured (bio)molecule by chromato-
din supports have been extensively used in affinity-based graphic and spectroscopic methods when it is released.
chromatographic strategies (Wilchek and Bayer, 1990). This
In this report, we describe our test system, the biotin/strepta-
method’s utility has been diminished by the very inefficient vidin complex in which the protein of interest is attached to the
release of the intact biotinylated protein from the streptavidin biotin by an acylhydrazone cleavable linker (Figure 1). Acylhydra-
support under mild conditions (Holmberg et al., 2005; Marie zones have been used extensively in drug-delivery systems
Chemistry & Biology 16, 763–772, July 31, 2009 ª2009 Elsevier Ltd All rights reserved 763

Chemistry & Biology
Proteomic Tools: Acylhydrazone Cleavable Linker
Figure 1. General Strategy of Cleavable Linker for Isolation, Identification and Detection of Target Proteins
(Sawant et al., 2006; Kale and Torchilin, 2007), in monitoring providing (bio)molecules that can be identified in proteomic
conjugation reactions (Flinn et al., 2004), and as structural units searches by conventional analytical techniques. We expect
in the construction of dynamic combinatorial libraries (Goral that incorporation of an acylhydrazone unit within a linker will
et al., 2001). Studies show that in most instances, acylhydra- be generalized, allowing for the selective release of tagged mole-
zones are stable under near-neutral to basic pH conditions (pH cules from noncovalently or covalently bound supports.
ꢁ8–10), but they undergo both hydrolysis (Flinn et al., 2004;
Smith et al., 2007) and hydrazone exchange with hydrazides RESULTS
(King et al., 1986) in moderately acidic solutions (pH ꢁ4–5).
Herein, we show that biotinylated probes with an imbedded acyl- Design and Chemical Properties of Cleavable Linker
hydrazone unit are readily cleaved in high yields at near-neutral Compound 1 was selected and synthesized as our test linker
pH values with acylhydrazides that contain a traceable tag (Figure 2). It contained an acylhydrazone cleavage site, a biotin
Figure 2. Synthesis of Acylhydrazone Linker 1 and Exchange of Hydrazone Linkage in 1 with Acethydrazide (7)
764 Chemistry & Biology 16, 763–772, July 31, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Proteomic Tools: Acylhydrazone Cleavable Linker
unit, a terminal azide on 1 to permit capture with an alkyne-modi-
fied protein using a copper(I)-mediated cycloaddition reaction
(‘‘click chemistry’’) (Agard et al., 2006; Dieterich et al., 2007;
MacKinnon et al., 2007; Rostovtsev et al., 2002; Speers et al.,
2003; Tornoe et al., 2002), and a polyethylene glycol linker to
increase its water solubility and to minimize adverse steric inter-
actions with the immobilized streptavidin during protein capture
(Marie et al., 1990). The success of 1 to capture biomolecules
hinged upon its ability to undergo Cu(I)-mediated cycloaddition
without disrupting the acylhydrazone linkage, its stability in
aqueous solutions, and its ability to undergo cleavage with acyl-
hydrazides. We first showed that the acylhydrazone linkage in 1
was stable to Cu(I)-mediated cycloaddition conditions (see
Figure S1 available online), and remained intact at pH 7.4 and
above but at pH 5.8 underwent consumption (38%) at 22ꢂC
(2 hr) (Table S1). Significantly, 1 efficiently reacted with acethy-
drazide (7) (20–100 Eq) at 22ꢂC to give 8 (Figure 2). For example,
at pH 5.2 and 6.2, the reaction was essentially complete within
1 hr at 22ꢂC, using as little as 20 Eq 7 (high-performance liquid
chromatography [HPLC] analysis) (Table S2). The ease of this
imine exchange was markedly faster than that reported by
King and coworkers for a similar transformation (King et al.,
1986). Next, we examined the stability of the acylhydrazone
linker in the presence of detergents (SDS, NP-40, Triton X-100)
because many proteomic protocols employ these agents to
facilitate protein solubilization. HPLC analysis showed that
when SDS (0.5% and 1%) was included in the buffered solutions
(pH 7.4) there was a rapid loss of 1, whereas similar losses were
not observed with the detergents NP-40 (1%) and Triton X-100
(1%) (Table S3).
Figure 3. Comparison of the Efficiency of Protein Release from BSA
Adducts (9 and 11) Modified with Either Cleavable Linker 1 or Non-
cleavable Linker 10
(A) Chemical structure of noncleavable linker 10 and two different BSA
samples (9 and 11) modified with either cleavable linker 1 or noncleavable
linker 10.
(B) All reactions were run in aqueous 50 mM HEPES (pH 5.8) using optimized
condition (7 [10 mM] and SDS [20 mM] at 50ꢂC [1 hr]) (Figure S5B). Lanes
marked ‘‘s’’ correspond to the supernatant removed from the bead mixture
after the initial treatment, whereas the lanes marked ‘‘b’’ refer to the sample
obtained after the beads were washed and then treated with loading buffer
and heated at 95ꢂC for either 5 min (Ab) or 15 min (lane Bb). For reaction B,
5- to 10-fold sample volumes from the recovered Bb were loaded to facilitate
comparison. The proteins were visualized by silver staining.
Use of 1 for Protein Capture, Release, and Detection
To evaluate the utility of 1 for protein capture and release, we
prepared bovine serum albumin (BSA) sample 9 (proposed
general structure of the photoadduct; the structure of the azide
photoadduct has not been determined) (Figure 3A) after photo-
crosslinking with photoactivable lacosamide probe followed by mined from the ratio of the amount eluted under mild cleavage
click chemistry with 1 (Figure S2). The BSA sample 9 was conditions to that captured by streptavidin prior to cleavage.
captured by streptavidin-agarose beads and divided into equal Significantly, more than 85% of the protein captured by strepta-
aliquots. Each portion was either treated with or without 7 (50, vidin-agarose beads was released with 7 using mild conditions
200 mM) at different pH values (5.8, 7.4) to release the captured (7 [100 mM], SDS [20 mM] in aqueous 50 mM HEPES [pH 5.8]
proteins. We also tested whether either p-anisidine or aniline [50ꢂC, 1 hr]). Under harsh cleavage conditions (SDS loading
would facilitate acylhydrazone cleavage because these aryl buffer [2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bro-
amines have been reported to catalyze oxime ligation, hydra- mophenol blue] [95ꢂC, 5 min]), protein recovery was near-quan-
zone formation, and transimination (acylhydrazone exchange) titative (see Supplemental Experimental Procedures). These
at moderate pH values (Dirksen et al., 2006a, 2006b). Consistent harsh conditions are expected to completely cleave the acyl
with our HPLC findings (Tables S1 and S2), we observed hydrazone bond.
increased linker cleavage at pH 5.8 compared with at pH 7.4,
Because SDS can affect protein structure, function, and mass
upon the addition of 7, and upon the inclusion of either p-anisi- spectrometric identification, we identified conditions for the
dine or aniline with 7 (Figure S3). We then examined the effect protein recovery step that excluded SDS as well as those that
of 20 mM SDS (0.5%) on the cleavage reaction of 9 in aqueous contained 20 mM SDS (0.5%) (Figure S5). We found that 7-medi-
pH 5.8 solutions at 22ꢂC. In agreement with our HPLC studies ated cleavage of streptavidin bound 9 led to high protein
(Table S3), we found that 20 mM SDS facilitated linker cleavage, recovery rates after 4 hr in pH 5.8 and pH 7.4 (37ꢂC) solutions
and that inclusion of both SDS and 7 provided slightly enhanced without 20 mM SDS. Specifically, we observed that at pH 5.8,
levels of cleavage than with either one alone (Figure S4).
inclusion of p-anisidine in the reaction solution led to efficient
To examine the recovery efficiency of protein capture and cleavage (ꢁ97%) at 37ꢂC after 4 hr. Correspondingly, high
release of our method, we quantified the percentage yield of protein recovery rates (ꢁ93% to ꢁ98%) were also obtained at
the immobilization, flow-through/wash, and elution steps using 50ꢂC after 1 hr in acidic solutions (pH 4.2 and 5.8) upon the inclu-
model protein 9. The release efficiency could be readily deter- sion of 20 mM SDS with 7. Reducing the temperature from 50ꢂC
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Chemistry & Biology
Proteomic Tools: Acylhydrazone Cleavable Linker
Figure 4. Use of 1 for Adduct Capture and
Validation of the Release and Reduction
Steps
bead was treated with SDS-loading
buffer at 95ꢂC for 15 min (Figure 3B,
lane Bb). We estimated that the relative
efficiencies of protein recovery from the
cleavable linker BSA adduct 9 under
moderate conditions (pH 5.8, 7 [100 mM],
SDS [20 mM], 50ꢂC, 1 hr) compared with
the noncleavable linker BSA adduct 11
after heating (95ꢂC) with SDS-loading
buffer for 15 min to be ꢁ20:1, based on
comparison of the silver-stained protein
lanes in Figure 3B for As and Bb.
Structural Validation of the
Capture, Release, and Reduction
Steps and Detection of Protein
Using Traceable Tag
An important structural feature in the ac-
ylhydrazide-mediated cleavage of linker
1 is the formation of a new acylhydrazone
(e.g., 8). We anticipated that incorpo-
rating
a traceable acylhydrazone tag
(i.e., isotopic, radioactive, fluorescent) in
the captured (bio)molecule would facili-
tate its detection and identification. The
key chemical steps of the acylhydra-
zone-based cleavable linker method-
ology were validated by HPLC and mass
spectrometry beginning with N-acetyl-
L-cysteine methyl ester (12) (Figure 4).
(ꢁ93%) to 37ꢂC (ꢁ72%) to 22ꢂC (ꢁ48%) for the pH 5.8 reactions
led to progressively lower cleavage yields.
Reacting 12 with N-(propyl-2-ynyl)maleimide (13) gave 14 as
a mixture of diastereomers. Compound 14 was then treated
with 1 using CuBr-mediated cycloaddition conditions to provide
15. The 1H nuclear magnetic resonance (NMR), 13C NMR, and
mass spectral data were in agreement with the proposed adduct
(Figure S7A). Treatment of 15 with an ꢁ1:1 mixture of 7 and 7-d3
(p-anisidine [10 mM], pH 5.7, 37ꢂC, 2 hr) gave the corresponding
exchanged acetylhydrazone 16 and 16-d3 as the major product
(HPLC tR 12.8 min, Figure S7-B1). Mass spectral analysis of
the HPLC-purified 12.8 min product showed a diagnostic
‘‘doublet’’ at m/z 608.2 and 611.2 for 16 and 16-d3 [M + Na]+,
respectively (Figure S7-B2). Correspondingly, sequential conver-
sion of 15 to imine 16 and 16-d3 (pH 5.8) followed by NaCNBH3
reduction (pH 3.8) led to the appearance of a new peak in the
HPLC (Figure S7-C1). Isolation of the new product by HPLC
and mass spectral analysis gave a doublet pattern at m/z 610.2
and 613.2 consistent with the reduced acethydrazides 17 and
17-d3 [M+Na]+, respectively (Figures 4 and S7-C2). The facility
of this reduction is important because it provides a chemically
stable product not readily susceptible to acid hydrolysis and
which is suitable for mass spectral analysis. When the NaCNBH3
reduction was conducted at higher pH values (pH ꢁ7.4) 17 and
17-d3 were not observed but rather we isolated the anisidine
To investigate the efficiency of cleavable linker 1 for protein
capture and release compared with a noncleavable linker, we
synthesized linker 10 (Sun et al., 2006) (Figures 3A and S6). We
compared streptavidin-bound BSA 9 containing 1 with streptavi-
din-bound BSA 11 containing 10 (Figure 3A). Noncleavable linker
10 cannot undergo acylhydrazone cleavage, so protein release
can only occur by disruption of the biotin-(strept)avidin complex
(Holmberg et al., 2005; Jenne and Famulok, 1999; Tong and
Smith, 1992). Both streptavidin-bound BSA 9 and 11 were
treated for 1 hr at pH 5.8 using 7 (100 mM) and SDS (20 mM)
at 50ꢂC (Figure 3B). The extent of modified BSA recovery was
determined by analyzing the supernatant and bead samples.
Inspection of Figure 3B showed that very low levels of protein
recovery were obtained from the initial supernatant from the
BSA sample 11 modified with the noncleavable linker 10 (Fig-
ure 3B, lane Bs). Correspondingly, we observed high protein
recovery from the supernatant from BSA 9 containing the cleav-
able linker 1 (Figure 3B, lane As). Consistent with reports in the
literature (Holmberg et al., 2005; Tong and Smith, 1992), we
observed low levels of protein recovery from the streptavidin-
bound BSA 11 containing the noncleavable linker 10 after the
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Chemistry & Biology
Proteomic Tools: Acylhydrazone Cleavable Linker
Figure 5. Use of 1 for Enolase Capture, Release, and Detection
(A) Scheme of enolase capture, release, and detection. The single cysteine unit in enolase (19) was treated with 13 to give approximately 30% of alkyne function-
alized enolase 20 based on mass spectrometry (data not shown) and then converted to enolase 21 under Cu(I)-mediated cycloaddition condition.
(B) Use of fluorescein hydrazide 22 for detection and isolation of streptavidin-bound modified enolase 21. Lane 1, 23 (the enolase recovered after incubation of
enolase 21 with streptavidin beads in aqueous 50 mM HEPES (pH 5.8), followed by washing of the beads, and then treatment of the beads with 22 (1.5 mM) using
an optimized condition (p-anisidine [10 mM], 37ꢂC, 4 hr); lane 2, 24 (reduction of 23 with NaCNBH3 at pH 3.8); lane 3, control (all reactions were the same as 23
except the cleavable linker was excluded in the cycloaddition step). The proteins were visualized by fluorescent detection by excitation at 488 nm and detection at
520 nm.
derivative 18 (m/z 637.3 [M+H]+) (Figures 4 and S7-D2). This mouse soluble liver lysate. We asked whether the use of 25
finding suggested that at near-neutral pH values, 16 and 16-d3 and cleavable linker 1 would permit efficient capture and removal
underwent transimination with anisidine and then reduction to of ALDH-1 from the mouse soluble liver proteome using strepta-
give 18 as the major product. These results demonstrated that vidin beads, and whether the release of the captured ALDH-1
the acylhydrazide-mediated cleavage of the linker provided from the streptavidin support with 7 would proceed with minimal
a traceable tag in the cleaved product that can be monitored release of nonspecific ‘‘background’’ proteins that bound to the
by mass spectrometry.
Similarly, we showed that fluorescein hydrazide treatment of
streptavidin resin (Fonovic et al., 2007).
Accordingly, mouse soluble liver lysate (pH 8.0) was incubated
enolase sample 21 modified with cleavable linker 1 (Figure 5A) with 25 (5, 25 mM) at room temperature (1 hr) and then treated
led to the appearance of a strong fluorescent band correspond- with rhodamine azide (RhN3, 26), TCEP, TBTA, and CuSO4.
ing to the expected enolase adducts both before and after The lysate was separated by SDS-PAGE and the labeled
NaCNBH3 (pH 3.8) treatment (Figure 5B). Because only fluores- proteins visualized by in-gel fluorescence (Figure 6B). In agree-
cently labeled proteins are released, this methodology can ment with earlier reports (Speers and Cravatt, 2004), only a single
significantly increase both the detection sensitivity and purifica- protein band was observed at ꢁ50 kDa when 5 mM 25 was used
tion efficiency of the proteins of interest in proteomic analyses.
(Figure 3A, lane 1). Increasing the concentration of 25 to 25 mM
led to increased intensity of this band and the appearance of
lower molecular weight bands (Figure 6B, lane 3). The in-gel
detection of the ꢁ50 kDa band was consistent with the selective
Use of Cleavable Linker 1 in Proteomic Target Searches:
Capture and Identification
Next, we tested whether cleavable linker 1 can be applied to tagging of ALDH-1 by activity probe 25 as detailed in the litera-
capture proteins of interest from a natural proteome. The Cravatt ture (Speers and Cravatt, 2004).
laboratory showed that alkyne phenyl sulfonate ester 25 (Speers
Theexperimentwasrepeatedwith25(25mM)substituting either
and Cravatt, 2004; Weerapana et al., 2008) (Figure 6A) selec- cleavable linker 1 or noncleavable linker 10 for 26. The labeled
tively reacted with aldehyde dehydrogenase 1 (ALDH-1) in the complexes were captured by adding streptavidin-agarose beads.
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Chemistry & Biology
Proteomic Tools: Acylhydrazone Cleavable Linker
Figure 6. Proteomic Target Search in Mouse
Liver Proteome with the Cleavable Linker 1
Using 25 and Comparison with Noncleavable
Linker 10
(A) Chemical structures of 25 and 26.
(B) After labeling with 25 (5 mM or 25 mM) followed by
Cu(I)-mediated cycloaddition with 26, the signal was
detected by in-gel fluorescence scanning. Using
5 mM 25, ALDH-1 (arrow) was selectively labeled. At
25 mM 25, ALDH-1 (arrow) and an abundant protein(s)
(asterisk) in liver lysate were labeled.
(C) After labeling with 25 (25 mM), probe-labeled
proteins were captured by streptavidin beads after
Cu(I)-mediated cycloaddition to either cleavable linker
1 or noncleavable linker 10. The beads were washed
and treated using
a mild cleavage condition (7
[100 mM], p-anisidine [10 mM] in aqueous 50 mM
HEPES [pH 5.8] [37ꢂC, 4 hr]). Lanes marked ‘‘s’’ corre-
spond to the supernatant removed from the bead
mixture after the initial treatment, whereas the lanes
marked ‘‘b’’ refer to the sample obtained after treat-
ment of the remaining beads with loading buffer
(95ꢂC, 5 min). The proteins were visualized by silver
staining.
(D) Comparison of the release efficiency of 25-labeled
target protein (ALDH-1) after Cu(I)-mediated cycload-
dition to either cleavable linker 1 or noncleavable linker
10 by western blot using anti-ALDH-1 antibody.
After extensive wash with buffer, the recovered beads from both able linker 1 upon treatment with 7 but not in the corresponding
the biotinylated cleavable 1 and the noncleavable 10 probes supernatant from noncleavable linker 10 (Figure 6D, lane As
were treated with 7 (100 mM) and p-anisidine (10 mM), pH 5.8 versus lane Bs). Analysis of the SDS-loading buffer-treated
at 37ꢂC (4 hr), conditions designed to release proteins from beads from both reactions showed the presence of ALDH-1
cleavable linker 1. The supernatants from these reactions (Figure 6D, lanes Ab and Bb). The silver-stained and western
were recovered. The remaining beads were treated with SDS- blot gels documented that the supernatant recovered from the
loading buffer, heated at 95ꢂC (5 min), and the supernatants cleavable linker 1 after 7-mediated cleavage was highly en-
recovered. The recovered protein samples were loaded on riched with target protein ALDH-1 and was devoid of most
SDS-PAGE and the resolved bands visualized by silver staining background proteins present in the lysate. Correspondingly,
(Figure 6C) and analyzed by western blot (Figure 6D). The silver- ALDH-1 was not detected in the supernatant from the noncleav-
stained gel lane for the 7-treated supernatant from cleavable able linker 10 (Figures 6C and 6D, lane Bs), but it was observed
linker 1 was remarkably clean, displaying only two prominent after harshly heating (95ꢂC, 5 min) the recovered streptavidin
bands at ꢁ50 kDa and ꢁ44 kDa (Figure 6C, lane As). This result resin with SDS-loading buffer (Figures 6C and 6D, lane Bb),
is consistent with that shown in Figure 6B. Based on the result a condition that results in the release of many background
from Coomassie blue staining of the untreated whole lysate, proteins.
we suspect that the 44 kDa band corresponds to abundant
protein(s) (data not shown). Significantly, the lane from the DISCUSSION
supernatant recovered from the remaining beads treated with
loading buffer (95ꢂC, 5 min) showed these two bands along We selected 1 as our prototype acylhydrazone linker because of
with significant amounts of other bands (Figure 6C, lane Ab), its structural properties and its expected solubility in water. The
indicating that many background proteins are indeed nonspe- acylhydrazone moiety in 1 is the cleavage site, and it provided
cifically captured by the streptavidin-agarose beads. Corre- the necessary balance between stability and reactivity. We found
spondingly, the 7-treated supernatant from noncleavable linker that the acylhydrazone unit underwent cleavage under mildly
10 displayed only a weak band at ꢁ44 kDa and no band at acidic conditions (pH 5.8), but remained intact under near-
ꢁ50 kDa (Figure 6C, lane Bs), whereas the remaining beads neutral (pH 7.4) and basic (pH 10) conditions (Table S1). This
lane showed significant amounts of the ꢁ44 kDa and ꢁ50 kDa finding is in general agreement with earlier reports (Goral et al.,
bands corresponding to an abundant protein(s) and ALDH-1, 2001; King et al., 1986). The observed pH dependency for 1
respectively, and many other bands attributed to background hydrolysis led us to use moderately basic (pH 7.4–8.5) conditions
proteins (Figure 6C, lane Bb). To confirm that the selectively for storage, handling, and chemical transformations where
captured 50 kDa protein by the cleavable linker approach is cleavage of the linker was not desired. We identified additives
indeed ALDH-1, we performed western blot analysis using an that promoted cleavage of the acylhydrazone group. Using the
ALDH-1 antibody (Figure 6D). We observed a prominent band findings of Dirksen et al. (2006a, 2006b), we confirmed that
consistent for ALDH-1 in the supernatant recovered from cleav- both aniline and p-anisidine facilitated acylhydrazide exchange
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Proteomic Tools: Acylhydrazone Cleavable Linker
(pH 5.8, 7.4) (Figure S3). A surprising result was that the anionic Correspondingly, 7-mediated cleavage of 11 led to the release
detergent SDS fostered acylhydrazone cleavage (Figure S4 and of very low levels of BSA (Figure 3B). When we compared the rela-
Table S3). Correspondingly, use of nonanionic detergents, tive amounts of BSA recovered from streptavidin-bound BSA 9
NP-40 and Triton X-100, in place of SDS did not promote (containing cleavable linker 1) with streptavidin-bound BSA 11
cleavage. The chemical factors responsible for the SDS-medi- (containing noncleavable linker 10), we observed a ꢁ20-fold
ated cleavage have not been determined. Specifically, we do increase in BSA recovery with 9. This increased amount is signif-
not know whether SDS serves as a specific or general base in icant because BSA release from 9 proceeded under mild condi-
the hydrolysis step, or whether SDS-mediated acylhydrazone tions whereas harsh conditions were required for 11. Using the
loss is due to direct attack of the hydrazone linkage by SDS, or modified BSA protein 9, we showed that 7-mediated acyl hydra-
whether SDS-mediated loss is due, in part, to the formation of zone cleavage under mild conditions led to the release of more
micelles that facilitates hydrazone modification (Camilo and Pilli, than 85% of the protein captured by the streptavidin-agarose
2004; Gogoi et al., 2005; Wang et al., 2005). Most important for beads (see Supplemental Experimental Procedures).
our study was our demonstration that 7 induced hydrazone
The cleavage step was validated by treatment of the modified
exchange in 1. Acethydrazide exchange of acylhydrazones has N-acetyl-L-cysteine methyl ester adduct 15 with 7 and 7-d3
been described by King et al. (1986). The reaction was reported (Figure 4). Mass spectrometric analysis of both the cleaved ace-
to proceed to near completion within 2 days at pH 4.7 and 5.2 tylhydrazone adducts 16 and 16-d3, and the corresponding
(25ꢂC), but at pH 7.0 substantial amounts of unreacted starting NaCNBH3-reduced adducts 17 and 17-d3 showed a character-
material (ꢁ40%) remained after 8 days. We found that with 1 istic mass spectrometric signature pattern ([M] + [M+3]) for the
the reaction proceeded to near completion at pH 5.2 and 6.2 desired products (Figure S7). Our finding that NaCNBH3 reduc-
within 1 hr at 22ꢂC, and that high yields of exchange were tion of the reaction mixture containing 16, 7, and 7-d3 at pH
observed at pH 7.4 after 7 hr at 22ꢂC (Table S2). Similarly efficient ꢁ7.4 provided p-anisidine adduct 18 indicated that employment
cleavage for the acylhydrazone group was observed in the BSA- of a mixture of nondeuterated and deuterated aniline derivatives
modified product 9 bound to the streptavidin support, and the in the cleavage step can similarly provide a diagnostic mass
reaction was catalyzed by the additive p-anisidine, which spectral pattern useful for protein identification.
permitted efficient cleavage at pH 7.4 within 2 hr (Figure S3).
An important component in the design of the acylhydrazone
The facility with which 7 cleaves 1 at near-neutral pH values linker was incorporating a traceable tag upon protein release
compared with the earlier findings (King et al., 1986) suggests from the support. We further documented this design feature
that structural changes surrounding the acylhydrazone linkage using fluorescent hydrazide 22 for acylhydrazone linker cleavage
affect cleavage rates.
in streptavidin-bound enolase 21 (Figure 5). With 22 we showed
To test the utility of 7-mediated cleavage of acylhydrazone that acylhydrazone exchange of streptavidin-bound enolase 21
linkers for protein capture and isolation, we used two protein- led to a protein band in the gel that was readily visualized on fluo-
based model systems. Because linker 1 contained a terminal rescent imaging. Because only the tagged proteins were fluores-
azide unit, we installed an alkyne moiety (moieties) within the cently labeled and released, this approach could significantly
test proteins to permit Cu(I)-mediated cycloaddition reaction. increase both the detection sensitivity and purification efficiency
Accordingly, BSA was randomly modified with the photoaffinity of the proteins of interest in proteomic analyses. Finally, we
lacosamide probe to give an alkyne functionalized BSA (Fig- recognized that the acylhydrazone group in the protein-released
ure S2). Correspondingly, the single cysteine residue in S. cerevi- product can undergo reduction with NaCNBH3 to the corre-
siae enolase (19) (Chin et al., 1981; Holland et al., 1981) permitted sponding acylhydrazide, a chemically stable product not readily
the Michael addition to N-(prop-2-ynyl)maleimide (13) to give susceptible to acid hydrolysis. Thus, we reduced the hydrazone
alkyne functionalized enolase 20 (Figure 5). Together these modi- group in enolase 23 with NaCNBH3 in acid to give 24. This reduc-
fied proteins permitted us to test the utility of acylhydrazone linker tion step provides a convenient opportunity to incorporate
1. The biotinylated BSA products 9 and 11 obtained after Cu(I)- a radioactive tag in the protein through the use of [3H]-NaCNBH3,
mediatedcycloaddition with 1 and 10, respectively, were captured which can aid protein detection and identification studies.
with streptavidin beads. Utilizing the information learned in our
The utility of cleavable linker 1 was tested in a proteomic
model studies with 1, we identified a series of conditions that search. Cravatt and coworkers have elegantly demonstrated
led to efficient cleavage of the acylhydrazone linker in BSA 9, that ALDH-1 was selectively labeled in the mouse soluble liver
allowing protein release from the streptavidin support. Using lysate by phenyl sulfonate ester 25 at pH 8.0. The specificity of
100 mM 7 and 20 mM SDS, we observed efficient cleavage and this activity probe for ALDH-1 provided a stringent test for our
release of modified BSA within 1 hr at 50ꢂC in pH 5.8 solutions linker. We asked if linker 1 selectively reacted with 25-modified
(Figure S5). High yields of modified BSA release were also ALDH-1 by Cu(I)-mediated cycloaddition and whether, upon
observed within 4 hr in both pH 5.8 (22ꢂC) and pH 7.4 (37ꢂC) solu- streptavidin bead capture and treatment of the resin with 7 under
tions containing 7 (100 mM) and p-anisidine (10 mM). Excluding mild conditions, the supernatant would be enriched in ALDH-1
SDS in the cleavage protocol might facilitate subsequent mass and be devoid of background proteins. Using cleavable linker
spectrometric detection of the recovered protein and permit us 1, we demonstrated that the supernatant recovered after treating
to identify multiprotein complexes that maintain their bioactive the streptavidin beads with 7 under mild conditions was enriched
conformations. Finally, we expect that the acylhydrazone group in ALDH-1 and was remarkably clean of background proteins
generated after linker cleavage can serve as a convenient alde- (Figure 6C). The silver-stained gel for this sample showed only
hyde-protecting group that can be removed with mild acid, two prominent protein bands, the first at ꢁ50 kDa corresponding
thereby permitting further protein tagging and modification. to ALDH-1 and the second at ꢁ44 kDa, which we suspect is an
Chemistry & Biology 16, 763–772, July 31, 2009 ª2009 Elsevier Ltd All rights reserved 769

Chemistry & Biology
Proteomic Tools: Acylhydrazone Cleavable Linker
abundant protein(s) in the lysate that was modified by the 25 mM
Our procedure compliments the widely used method that
25. The absence of background proteins in Figure 6C, lane As, is couples on-bead digestion and mass spectrometry (Chresten-
important because proteomic target identification studies have sen et al., 2004). This on-bead method benefits from its simplicity
been hampered by the presence of such proteins in samples and ease of use. Unlike the on-bead method, our procedure
containing the target(s) (Fonovic et al., 2007). As anticipated, allows the chemoselective release of the protein of interest, the
ALDH-1 was not detected in the supernatant after treatment of incorporation of an isotopic tag that facilitates mass spectro-
the beads with 7 when the noncleavable linker 10 was employed metric identification, the isolation of the intact protein, and the
in the Cu(I)-mediated cycloaddition reaction, but it was observed opportunity to identify the site of protein adduction.
after harshly heating (95ꢂC, 5 min) the recovered streptavidin
resin with loading buffer containing 2% SDS and 1% mercaptoe-
thanol, a condition that results in the release of many back-
ground proteins that nonspecifically bound to streptavidin resin.
The recovered yield for ALDH-1 in the supernatant was some-
what lower than that observed for cleaved BSA 9 under similar
conditions (Figures 6 and S5-A), and might reflect the different
nonspecific matrix binding properties of these two proteins.
Ourstudies demonstrated that incorporating an acylhydrazone
group within the biotin-containing linker permitted efficient linker
cleavage from the streptavidin support. Some advantages asso-
ciated with this method include: (1) the ease of linker synthesis; (2)
stability of the linker to bio-orthogonal coupling transformations
(e.g., Cu(I)-mediated cycloaddition reactions); (3) linker stability
at near neutral and moderately basic pH values; (4) chemoselec-
tive cleavage of the linker with acylhydrazides with minimal
release of background proteins nonspecifically bound to (strep-
tavidin) supports; (5) high recovery yields of the cleaved proteins;
and (6) ability to incorporate a traceable tag (e.g., isotopic, fluo-
rescent) in the captured protein to facilitate detection and identi-
fication. Finally, the mild conditions needed for acylhydrazone
cleavage offer the possibility that nondenatured proteins or
protein complexes can be captured and then released. Coun-
tering these advantages is the need to avoid moderately acidic
pH values during protein capture to prevent premature linker
cleavage and the recognition that linker cleavage leads to
a permanent structural modification in the protein.
SIGNIFICANCE
In this study, we showed that the use of 1 permitted the rapid,
selective release of proteins from streptavidin supports with
acylhydrazides under mild conditions at near-neutral pH
values. Protein release proceeded with significantly reduced
levels of background proteins. The lack of appreciable
amounts of background proteins is important because pro-
teomic target identification studies have been hampered by
the presence of such proteins in samples containing the
target(s). In addition, the acylhydrazone group permitted
the incorporation of a traceable tag (i.e., isotopic, fluores-
cent) in the released protein. The use of tailored acylhydra-
zides that contain a traceable tag provides opportunities to
detect and identify biomolecules. We anticipate that the acyl-
hydrazone group will serve as a useful, cleavable structural
entity for the release of tethered molecules from supports
other than (strept)avidin (e.g., noncovalent complexes and
systems/resins) that employ a covalent linkage for (bio)-
molecule attachment to the support.
EXPERIMENTAL PROCEDURES
Synthesis of Compounds 3, 4, 7-d3, 8, 10, 13–15, 16, 16-d3, 17,
and 17-d3
See Supplemental Data.
The acylhydrazone linker compares favorably with previously
introduced cleavable linkers. Like 1, each linker has advantages
and disadvantages to consider prior to selection and use. For
instance, photolytic-based cleavable linkers typically undergo
efficient release, but upon cleavage, they release reactive
carbonyl compounds (aldehydes, ketones) that can react with
the protein (Bai et al., 2004; Thiele and Fahrenholz, 1994).
Acid-sensitive linkers that require trifluoroacetic acid (TFA) can
lead to nonspecific cleavage, and TFA use requires its removal
prior to mass spectrometry analysis (van der Veken et al.,
2005). Linkers that rely on proteolytic enzymes (e.g., trypsin)
provide a selective cleavage method but require that a designed
peptide sequence be installed within the linker that is efficiently
cleaved (Dieterich et al., 2007; Fonovic et al., 2007; Speers and
Cravatt, 2005). The use of proteolytic enzymes for cleavage
may prevent the isolation of the intact protein. Proteomic exper-
iments utilizing disulfide linkers require the use of buffers that are
devoid of reducing reagents (Finn et al., 1985; Fonovic et al.,
2007; Marie et al., 1990; Shimkus et al., 1985). Moreover, these
disulfide linkers can be cleaved under intracellular conditions.
This concern for premature cleavage of the linker is not an issue
for the diazobenzene linker advanced by Bogyo and coworkers
and where Na2S2O4 serves as the chemoselective reductant
(Fonovic et al., 2007; Verhelst et al., 2007).
Preparation of N0-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)
acetaldehyde Biotinylhydrazone (1)
To an anhydrous CH2Cl2 solution (2 ml) of oxalyl chloride (0.10 ml, 1.10 mmol)
maintained at ꢀ78ꢂC under Ar was slowly added freshly distilled DMSO
(0.19 ml, 3.29 mmol). The mixture was stirred (30 min), and then dry 4
(0.20 g, 0.91 mmol) was added via syringe. The mixture was stirred for an addi-
tional 30 min at ꢀ78ꢂC, and then Et3N (0.78 ml, 6.57 mmol) was added (10 min)
and the mixture was stirred (15 min) at this temperature and warmed to
ambient temperature. The reaction solution was diluted with CH2Cl2 (10 ml)
and washed with H2O (2 3 10 ml). The organic layer was dried (Na2SO4),
concentrated, and the crude aldehyde 5 (0.19 g, 0.88 mmol) was dissolved
in CH2Cl2 (1 ml) and added to a solution of 6 (0.15 g, 0.58 mmol) in THF/H2O
(1:1, 5 ml). The reaction mixture was allowed to stir at room temperature
(16 hr), the solvent was evaporated in vacuo, and then the crude product puri-
fied by column chromatography (SiO2; 1/9 MeOH/CHCl3) to yield 0.20 g (74%)
of 1 as a white solid: mp 134ꢂC–135ꢂC; Rf = 0.35 (1/9 MeOH/CHCl3); IR (nujol)
2924, 2101, 1667, 1557, 1459 cmꢀ1 1H NMR (CD3OD) (minor conformer in
;
parenthesis [a mixture of conformers similar to other hydrazones (Syakaev
et al., 2006)]) d 1.45–1.52 (m, C(6)H2), 1.58–1.79 (m, C(7)H2, C(8)H2), 2.27
(t, J = 7.4 Hz, C(9)H2 (2.65 (t, J = 7.5 Hz, C(9)H2))), 2.71 (d, J = 12.6 Hz,
C(5)HH0), 2.93 (dd, J = 4.7, 12.6 Hz, C(5)HH0), 3.18–3.25 (m, C(2)H), 3.37
(t, J = 4.8 Hz, N3CH2), 3.65–3.69 (m, 2 OCH2CH2O, N3CH2CH2), 4.18 (d, J =
5.3 Hz, OCH2CHN (4.15 (d, J = 5.3 Hz, OCH2CHN))), 4.31 (dd, J = 4.2,
7.6 Hz, C(3)H), 4.49 (dd, J = 4.7, 7.6 Hz, C(4)H), 7.49 (t, J = 5.3 Hz, OCH2CHN
(7.33 (t, J = 5.3 Hz, OCH2CHN))); 13C NMR (CD3OD) (minor conformer in paren-
thesis) d 26.0 (26.6) (C(6)), 29.6 (C(7)), 29.9 (30.0) (C(8)), 33.2 (35.2) (C(9)), 41.2
(C(5)), 51.9 (N3CH2), 57.1 (57.2) (C(2)), 61.8 (C(3)), 63.5 (C(4)), 71.2, 71.3, 71.4,
770 Chemistry & Biology 16, 763–772, July 31, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Proteomic Tools: Acylhydrazone Cleavable Linker
71.5, 71.7, 71.8 (3 CH2OCH2), 145.6 (149.3) (OCH2CHN), 166.3 (C(20)), 172.7
(177.9) (C(10)); HRMS (ESI) 458.2182 [M + H+] (calcd. for C18H32N7O5S
458.2186); Anal. (C18H31N7O5S, 0.25 H2O) calcd.: C, 46.79%; H, 6.87%;
N, 21.22%; S, 6.94%. Found: C, 46.67%; H, 6.82%; N, 20.88%; S, 6.88%.
solution (10 ml) of either 1 or 10 (22 mg, 50 nmol) in 5% CH3CN/aqueous 50
mM HEPES (pH 7.4), a 5 mM solution (10 ml) of biotin hydrazide (6) (13 mg,
50 nmol) in CH3CN/aqueous 50 mM HEPES (pH 7.4), and CuBr (0.2 mg,
1.4 mmol). Using method B, immobilized streptavidin slurry (300 ml) was added
and the beads were treated with 7 (0.78 mg, 10 mmol) and p-anisidine [final
concentration 10 mM] at 37ꢂC (4 hr). The supernatants were collected and
then the remaining streptavidin beads were boiled (5 min) with SDS-PAGE
loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromo-
phenol blue). The samples were loaded on two SDS-PAGE (10%) gels and
the proteins visualized by silver staining and ALDH-1 detected by western blot
using anti-ALDH-1 antibody (ab23375, Abcam) as detailed in Supplemental
Data.
Treatment of Enolase (19) with Maleimide 13 to Afford Enolase 20
To a 200 mM solution (1 ml) of S. cerevisiae enolase (19) (Sigma, E6126) in
aqueous 50 mM HEPES (pH 7.4) was added a 200 mM solution (0.1 ml) of
13 (2.7 mg, 20.0 mmol) in 5% CH3CN/aqueous 50 mM HEPES (pH 7.4). The
solution was incubated at room temperature (2 hr) and then the reaction solu-
tion was diluted with aqueous 50 mM HEPES buffer (pH 7.4) to 5 ml and
passed through NAP-5 columns pre-equilibrated with 50 mM HEPES buffer
(pH 7.4). The eluents (ꢁ10 ml) were combined and stored at 4ꢂC.
SUPPLEMENTAL DATA
Cycloaddition Reaction between Enolase 20 and Cleavable Linker 1
to Afford Enolase 21 (Method A)
Supplemental Data include Supplemental Experimental Procedures, eight
figures, and three tables and can be found with this article online at http://
www.cell.com/chemistry-biology/supplemental/S1074-5521(09)00184-7.
To an aqueous 50 mM HEPES solution containing 20 obtained after passage
through the NAP-5 column (1 ml), was sequentially added a 20 mM solution
(50 ml) of 1 (0.44 mg, 1.0 mmol) in 5% CH3CN/aqueous 50 mM HEPES (pH 7.4),
a 20 mM solution (50 ml) of biotin hydrazide (6) (0.26 mg, 1.0 mmol) in 5%
CH3CN/aqueous 50 mM HEPES (pH 7.4), and CuBr (0.2 mg, 1.4 mmol). The
reaction mixture was rotated using Roto-shake (8 rpm, Scientific Industries
Inc., model SI-1100, Bohemia, NY) at room temperature (1 hr), then divided
in two equal portions and passed through separate NAP-5 columns pre-equil-
ibrated with HEPES buffer (pH 7.4) to give an aqueous solution of enolase 21.
The eluents were combined (ꢁ2 ml) and stored at 4ꢂC.
ACKNOWLEDGMENTS
We thank Steven Cotten (University of North Carolina-Chapel Hill) for helpful
discussions and advice. The project described was supported by grant
5R01NS054112 (to H.K., R.L.) from the National Institute of Neurological Disor-
ders and Stroke, and by a Korean Research Foundation Grant funded by the
Korean Government (MOEHRD, grant KRF-2006-352-C00042, to K.D.P.).
Use of Fluorescent Hydrazide 22 for Detection and Isolation
of Streptavidin-Bound Modified Enolase 21 and Reduction of Imine
23 to 24 (Method B)
Received: March 26, 2009
Revised: May 29, 2009
Accepted: June 5, 2009
Published: July 30, 2009
An aliquot of enolase 21 (200 ml) in aqueous 50 mM HEPES buffer (pH 7.4) was
added to an immobilized streptavidin slurry (1 ml) (High Capacity Streptavidin
Agarose Resin, Pierce, Rockford, IL) and rotated using a shaker (15 rpm) at 4ꢂC
(90 min). The streptavidin beads were washed with aqueous 15 mM HEPES
buffer (pH 7.4) (10 3 0.8 ml). The beads were centrifuged (1000 rpm, 1 min),
and the supernatant removed. An aqueous 50 mM HEPES solution (100 ml,
pH 5.8) was added and the beads treated with fluorescent hydrazide 22 (Alexa
Fluor hydrazide, A10436, Molecular Probes, 85.5 mg, 0.15 mmol; final concen-
tration 1.5 mM) and p-anisidine (123.0 mg, 1 mmol; final concentration 10 mM).
The reaction mixture was gently shaken at 37ꢂC (4 hr) and the supernatant
(100 ml) containing 23 was collected.
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