Journal of the American Chemical Society
Communication
ization of the S-nitrosylated protein during protein enrichment
and purification as well as SDS-PAGE in-gel analysis, as shown
in Figure 4B. Again, mild photocleavage avoided the use of a
harsh, high-concentration reductant such as dithiothreitol or
mercaptoethanol as an exogenous elution agent, as in the
previous isolation methodologies, which might induce
fictitiously reduced disulfide bonds in the targeted protein
during the elution. SDS-PAGE of the photolyzed supernatant
(Figure 4B and Figure S7) indicated that only the human
serum albumin (HSA, 66kD) of all blood proteins can be S-
nitrosylated, confirming a previous report that SNO-albumin in
plasma serves as a stable circulating storage form of NO.10
Importantly, avidin−biotin affinity capturing introduces a
specific coumarin tag to the S-nitrosylation site after photo-
cleavage. This tagging feature allows the direct determination of
the residue that received SNO modification using mass
spectrometric localization of the modified cysteine. Accord-
ingly, nano-flow LC−MS/MS analysis of the peptides was
carried out after in-gel trypsinolysis of the HSA band. In Figure
S8, the triply charged precursor ion with four isotopic peaks
corresponds to the peptide ALVLIAFAQYLQQCcoumarin
PFEDHVK with the coumarin adduct (Δm/z = 342.1),
suggesting that the S-nitrosylation occurred on sequence
T21−41 of HSA. As revealed by the tandem MS/MS of the
extracted ion on the [M + H]3+ peak (m/z 926.1) in Figure 4C,
ACKNOWLEDGMENTS
■
This research was supported by NSFC (21173078, 20903039),
Shanghai Rising-Star Program (10QA1401600), Shanghai
Science and Technology Commission (11JC1403300), and
the Fundamental Research Funds for the Central Universities.
REFERENCES
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+
the Δm/z range between the two informative fragment ions y9
2+
and y7 corresponds to m/z 591.2 [glutamine (Q) + cysteine
(C) + coumarin adduct − 2H2O], which unambiguously
identifies the SNO site in HSA as the Cys-34 residue. These
results demonstrate that our new phototrigger has the desired
superiority in thiol-proteomics studies because it serves as an
efficient fluorescent marker for visualization of the target
proteins, provides photocleavage for affinity purification, and
introduces an identification tag for determining the specific
SNO site in the protein by mass analysis.
In summary, we have successfully constructed a new class of
target-activated phototriggers. Target binding sends a signal to
unlock the phototrigger, thus causing specific photocleavage.
The use of maleimide enabled the phototrigger to target
mercaptans selectively and efficiently in both small molecules
and large biomacromolecules. The locking and unlocking of the
phototrigger were used to demonstrate fluorescence detection
of thiol-bearing proteins, selective capture, target-activated
release, and tag conservation for mass identification. The new
concept of target-activated phototriggers offers a powerful tool
for significant applications in thiol-proteomics research, such as
protein cysteine S-nitrosylation.
ASSOCIATED CONTENT
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S
* Supporting Information
Preparation and experimental details (Figures S1−S8 and Table
S1) and complete ref 8b. This material is available free of
AUTHOR INFORMATION
■
Corresponding Author
Notes
The authors declare no competing financial interest.
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dx.doi.org/10.1021/ja300475k | J. Am. Chem. Soc. 2012, 134, 5052−5055