Isotope-coded, fluorous photoaffinity labeling reagents

Article abstract of DOI:10.1039/c2cc00027j

A pair of isotope-coded, fluorous photoaffinity labeling reagents has been developed and coupled with a peptide. The modified peptides form adducts with methanol upon light illumination, which show characteristic isotope labeling patterns in mass spectra and can be separated from other peptides through fluorous silica. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc00027j

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Isotope-coded, fluorous photoaffinity labeling reagentsw
Zhiquan Song, Weigang Huang and Qisheng Zhang*
Received 2nd January 2012, Accepted 14th February 2012
DOI: 10.1039/c2cc00027j
A pair of isotope-coded, fluorous photoaffinity labeling reagents has
been developed and coupled with a peptide. The modified peptides
form adducts with methanol upon light illumination, which show
characteristic isotope labeling patterns in mass spectra and can be
separated from other peptides through fluorous silica.
incorporated into proteins through metabolic interference after
several cell doublings. Equal amounts of the heavy isotope-
labeled proteome and the normal proteome are then mixed for
analysis. Because the intensities of the normal and heavy
isotope-labeled peptides correlate with the corresponding protein
abundance ratios, SILAC has been used to quantify in vivo
changes of different proteins in the proteome.10 Recently, SILAC
has also been used in combination with biochemical pulldown
experiments to identify cellular targets of small molecule
probes.11 However, a potential problem with SILAC is that
the cells need to be efficiently labeled through metabolic inter-
ference, which typically takes several cell doublings (1–2 weeks
for mammalian cells).
Identifying cellular targets of bioactive small molecules from
endogenous and synthetic chemical libraries is essential for
their further applications as drug candidates or chemical
probes.1 Various techniques, including affinity-based chemical
proteomics,2 genetics-based cDNA complementation and
RNA interference (RNAi),3 and transcriptional profiling-
based approaches4 have accordingly been developed. Among
these techniques, the affinity-based chemical proteomics is the
most popular strategy for target identification because the
entire proteome can be scanned without bias for the tightest
binder. However, the biochemical pulldown experiments typically
rely on the reversible interactions between ligands and their
targets. Consequently, it remains challenging when the target is
a low-abundant protein, or the small molecule–protein interaction
is weak, or the target protein is one member of a family of
proteins with similar functions. Moreover, it is difficult to distin-
guish the non-specific small molecule binding proteins from the
context-specific targets.
We have previously developed fluorous photoaffinity labeling
reagents in efforts to capture low affinity, low abundant or
transient proteins through light induced crosslinking of small
molecules with their interacting proteins.12 The fluorous tags are
introduced to enrich proteins with low abundance through
fluorous solid phase extraction (FSPE) over fluorous silica,13
while the diazirine is incorporated to label interacting partners
through covalent interactions upon light illumination. However,
the photoaffinity labeling has the tendency to crosslink with
nonspecific targets as well. Coupling the isotope labeling strategy
with fluorous photoaffinity approach could minimize the non-
specific interactions, and enhance the chances to identify cellular
targets of small molecule probes. Instead of tagging the proteins
with isotope-labeled tags, we chose to develop reagents that are
isotope-coded for quantitative proteomic analysis to identify
interacting proteins. As shown in Fig. 1A, both normal and
heavy isotope-labeled fluorous diazirines are attached to the
small molecule (SM) of interest. Equal amounts of the attached
probes will be used to treat cells, one with cell state 1 while the
Isotope labeling of partial or entire proteome for protein
profiling has gained wide popularity because of its quantification
accuracy and potential to distinguish real from nonspecific
interactions.5 The isotopes can be introduced through either
chemical or metabolic labeling methods. In chemical methods,
isotopes are introduced through tags that specifically react with
functional groups in proteins. The first such isotope-coded
affinity tag (ICAT) reacts with the sulfhydryl groups of cysteine
residues.6 Other commonly used tags for peptide or protein
labeling include isobaric tags for relative and absolute quantifi-
cation (iTRAQ)7 and tandem mass tags (TMT);8 both react
specifically with primary amine groups in the N-termini or the
side chains of lysine residues. In metabolic labeling approaches,
the most widely used method is stable isotope labeling with
amino acids in cell culture (SILAC).9 Briefly, the heavy isotope-
labeled amino acids are added to the cell culture medium and
Division of Chemical Biology and Medicinal Chemistry,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599,
USA. E-mail: qszhang@unc.edu
w Electronic supplementary information (ESI) available: Experimental
protocols, NMR spectra of key compounds, and Fig. S1. See DOI:
10.1039/c2cc00027j
Fig. 1 Fluorous isotope-coded affinity labeling reagents. (A) ICAT
reagents are used for quantifying protein levels in 2 cellular states.
(B) Chemical structures of the ICAT reagents.
c
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Scheme 1 Synthesis of deuterated 5-D4.
other with cell state 2. Light illumination will initiate crosslinking
of the tagged small molecule with its interacting proteins. The cell
lysates are then combined and digested with proteases to generate
peptides. The fluorous-tagged peptides are enriched by FSPE and
analyzed by liquid chromatography–mass spectrometry (LC-MS).
Because equal amounts of normal and heavy isotope-labeled small
molecules are used, the mass spectra of interacting proteins will
show isotope-labeling patterns (Fig. 1A), i.e., the intensities of the
M + H and M + D ions will correlate with the ratio of proteins
in the two states.
Fig. 2 The coupling of peptide RKRSRAE with 5-H4/5-D4 and
photolysis of the resulting conjugate. MS spectra of the reaction
mixtures that contain 7-H4/7-D4 (A) and 8-H4/8-D4 (B), respectively.
fluorous photoaffinity labeling reagents have photoaffinity
labeling capacity, and the resulting products can be separated
from other non-fluorous components in the mixture. Con-
sequently, we used the reaction mixture that contains 7-H4
and 7-D4 for the subsequent experiments.
The photolysis of the diazirine group is next investigated.
The peptide conjugates 7-H4 and 7-D4 were illuminated at
350 nm for 10 min and 254 nm for 5 min in MeOH. As shown
in the mass spectrum of the reaction mixture, the molecular
ion of the expected adducts with MeOH were obtained
(Fig. 2B). The tandem MS results of 8-H4 and 8-D4 were
consistent with this conclusion (Fig. S1, ESIw), further con-
firming that the photoaffinity labeling group was attached to
the lysine residue. Again, the products formed the isotope
labeling patterns, and the intensities of M and M + 4 ions
reflected the ratio of 5-H4 to 5-D4.
We chose 1-H4 and 1-D4 (Fig. 1B) as the first pair of
isotope-coded, fluorous photoaffinity labeling reagents to
develop because of our previous success in synthesizing 1-H4.12
The synthesis of 1-D4 starts with the commercially available
terephthalic-2,3,5,6-d4 acid 2. Activation of the acid with thionyl
chloride (SOCl2) followed by esterification with MeOH14
provided the monoester 3 in 54% yield. The carboxylic acid
group in 3 was then activated with 1,10-carbonyldiimidazole
(CDI) and subsequently reduced by sodium borohydride
(NaBH4)15 to generate alcohol 4 in 73% yield. Following the
literature protocol,12 the alcohol 4 was converted to the fluorous
deuterated diazirine 1-D4. Subsequent coupling of 1-D4 with N,N0-
disuccinimidyl carbonate (DSC) then provided 5-D4 (Scheme 1).
Similarly, 5-H4 was prepared from 1-H4 in an analogous manner.
The peptide RKRSRAE 6 serves as the substrate of protein
kinase G (PKG)16 and is selected as a model compound for
attaching to 5-H4 and 5-D4. To test whether the isotope-coded
pair can be used for quantification, a 1 : 1 mixture of 5-H4 and
5-D4 reacted with the peptide to generate the corresponding
conjugates 7-H4 and 7-D4. As shown in the mass spectrum of
the reaction mixture (Fig. 2A), the desired coupling products
were generated along with several unidentified species. The
structures of 7-H4 and 7-D4 were further confirmed by tandem
mass spectrometry analysis (Fig. S1, ESIw). As expected, the
isotope patterns of 7-H4 and 7-D4 were also observed in
Fig. 2A and the intensities of M (normal) and M + 4 (heavy
isotope-labeled) ions reflected the ratio of 5-H4 to 5-D4. The
molecular ion of the original peptide was also identified,
suggesting that the conversion of 6 to 7-H4 and 7-D4 was not
complete. We did not attempt to further improve the reaction
conversion or characterize the unknown species because the
goal of this work is to demonstrate that the isotope-coded,
To test the separation efficiency, the reaction mixture con-
taining 8-H4 and 8-D4 was mixed with the trypsin digest of
bovine serum albumin (BSA) to form a relatively complex
mixture with its mass spectrum shown in Fig. 3A. Compared
to other peptides in the mixture, the intensities of the fluorous
peptides are low. The mixture was then loaded on a column
with fluorous silica. Elution of the column with 60% methanol
in 10 mM ammonium formate removed the non-fluorous
peptides, while the fluorous peptides were obtained after
elution with methanol (Fig. 3B). These results demonstrated
that FSPE effectively enriched fluorous peptides from a
complex mixture. We also generated 8-H4/8-D4 from a 2 : 1
mixture of 5-H4/5-D4 and mixed it with the trypsin digest of
BSA (Fig. 3C) for FSPE. As expected, the fluorous peptides
were enriched after FSPE (Fig. 3D). Furthermore, the ratio of
the normal to deuterated fluorous peptides changed from 1 : 1
in Fig. 3B to approximate 2 : 1 in Fig. 3D, suggesting that the
intensities of the deuterated to non-deuterated peptides were
dictated by the ratio 5-H4/5-D4.
In summary, we have developed a novel pair of isotope-coded,
fluorous photoaffinity labeling reagents. The photoactive diazirine
group will capture the transient and/or weakly interacting
c
3340 Chem. Commun., 2012, 48, 3339–3341
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the isotope-coded, fluorous photoaffinity groups can also be
incorporated into drug candidates, oligonucleotides, or proteins
to identify novel small molecule–protein, protein–nucleotide, or
protein–protein interactions.
Notes and references
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Fig. 3 Separation of fluorous tagged peptides from a complex mixture.
The mass spectrum of a mixture that contains 8-H4/8-D4 (1 mL, derived
from 10 mM 6) and BSA digest (5 mL, 7.5 mM): (A) before FSPE, 8-H4/
8-D4 = 1 : 1; (B) after FSPE, 8-H4/8-D4 = 1 : 1; (C) before FSPE, 8-H4/
8-D4 = 2 : 1; (D) after FSPE, 8-H4/8-D4 = 2 : 1.
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proteins while the fluorous tag will enable the enrichment of
the labeled peptides. The isotope-coded information on the
new reagents could be used to quantify the interacting proteins
(like SILAC) but without the necessity to label the proteome
through metabolic interference (unlike SILAC). These features
will enable the reagents developed in this work suitable to capture
low abundance, low affinity, and context-specific interactions
in the cells. Although peptide is used for a demonstration,
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 3339–3341 3341