The azide−alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide−alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na_2S_2O_4, 2% HOCH_2CH_2SH, 10% HCO_2H, 95% CF_3CO_2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO_2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies

Bagert, John D.

Dieterich, Daniela C.

Hodas, Jennifer J. L.

Landgraf, Peter

Mahdavi, Alborz

Ngo, John T.

Schuman, Erin M.

Szychowski, Janek

Tirrell, David A.

English

Caltech Authors - Main

Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition

 S1 
Supporting Information 
Cleavable Biotin Probes for Labeling of 
Biomolecules via the Azide – Alkyne 
Cycloaddition 
Janek Szychowski
1
, Alborz Mahdavi
1
, Jennifer J. L. Hodas
2
, John D. Bagert
1
, John T. 
Ngo
1
, Peter Landgraf
3
, Daniela C. Dieterich
2,3
, Erin M. Schuman
2,4
 and David A. 
Tirrell
1
* 
*To whom correspondence should be addressed. Tel: 626-395-3140; Fax: 626-568-8743; 
E-mail: tirrell@caltech.edu 
 
 
 
 
Sequence and mass spectrum of protein 18; 
1
H NMR spectra for compounds 4, 5, 6, 9, 
11, 12 and 14a and 14b; 
13
C NMR spectra for compounds 4, 5, 6, 9, 12 and 14a and 14b; 
and MS/MS spectra of tryptic fragments are provided. 
 S2 
Protein 18 sequence. 
XRGSHHHHHHGSLSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATYGKI
TLKLICTTGKLPVPWPTLVTTCGYGVQCFARYPDHLKRHDFFKSAFPEGYVQER
TISFKDDGKFKTRAEVKFEGDTIVNRIKLKGIDFKEDGNILGHKLEYNYNSHDVYI
TADKQKTGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVRLPDNHYLLTQSV
ISKDPNEKRDHAVLHEFVTAAGITHGIDELYK 
 
 
 
Calculated MW when X = Hpg: 28091.6 (after protein maturation (loss of H2O and H2)
1
 
Calculated MW when X = Met: 28113.7 (after protein maturation (loss of H2O and H2)
1
 
Observed MW: 28092.0 
 
MS from 20000 to 31000 Da 
 
 
 
Expanded spectrum from 27900 to 28300 Da 
 
28092.0 
Note that there is no significant signal 
observed at 28113.7 Da 
 
28092.0 
28164.4 
 S3 
 
 
 
 
 
 
 
NO2
O
N3
4  
NO2
O
N3
4  
 S4 
 
 
 
 
 
 
 
 
 
5
NO2
OH
H2N
 
 
5
NO2
OH
H2N
 
 
5
NO2
OH
H2N
 
 S5 
 
 
 
 
 
 
 
 
 
 
 
NO2
O
NHHN
S
O
H
N
O
H H O
N
H
N
H
O
N3
6
 
 
 
 
NO2
O
NHHN
S
O
H
N
O
H H O
N
H
N
H
O
N3
6
 
 S6 
 
 
 
 
 
 
 
 
 
NHHN
S
O
H
N
O
H H O
N
H
OH
N
N
H
N
O
N3
9  
 
NHHN
S
O
H
N
O
H H O
N
H
OH
N
N
H
N
O
N3
9  
 S7 
 
 
11
NHHN
S
O
H
N
O
H H
O
4
O
N
H
OH
 
 S8 
 
 
 
 
 
 
 
 
 
 
 
 
12
NHHN
S
O
H
N
O
H H
O
4
O
N
H
O
Si
O
N3
Ph Ph
 
12
NHHN
S
O
H
N
O
H H
O
4
O
N
H
O
Si
O
N3
Ph Ph
 
 
12
NHHN
S
O
H
N
O
H H
O
4
O
N
H
O
Si
O
N3
Ph Ph
 
 S9 
 
 
 
 
 
 
 
 
 
 
 
 S10 
 
 
 
 
 
 
 S11 
 
Orientation of the click reaction. The relative efficiencies of labeling with probes 14a 
and 14b were determined by using EGFP purified from HEK 293T cells treated with Hpg 
(1) or Aha (21).  Conjugation of Aha- or Hpg-tagged proteins with their complementary 
alkyne or azide probes (14b or 14a, respectively) enabled comparison of the total amount 
of GFP with the level of biotinylated protein. As shown in Figure S1, Aha- and Hpg-
tagged proteins were labeled at levels that were indistinguishable. 
 
Figure S1. Hpg- and Aha-tagged EGFP were affinity-purified using the µMACS
TM
 GFP 
Tag Protein Isolation Kit.  Identical amounts of purified Aha- or Hpg-tagged proteins 
were reduced, alkylated and labeled with probe 14a or 14b. All samples were processed 
twice in parallel for western blot analysis; tagged EGFP was detected with antibodies 
directed against EGFP or Biotin. Labeling efficiencies for probes 14a and 14b are 
indistinguishable. 
 
 S12 
 
Expression of EGFP in HEK 293T cells supplemented with Hpg or Aha. HEK 293T 
cells were transfected with 10 µg pEGFP-N3 (Clontech Laboratories, Inc., 
Mountain View, CA, U.S.A.) plasmid DNA using Lipofect 2000 (Invitrogen, 
Karlsruhe, Germany) according to manufacturer’s instructions. Transfection 
efficiencies and expression rates were monitored by fluorescence microscopy. 
After 12 h the medium was exchanged with fresh DMEM + (-Met) and 
supplemented with either 4 mM Hpg or 4 mM Aha. After 24 h cells were 
harvested in 10 ml ice-cold TBS pH 7.4 (25 mM Tris-HCl pH 7.4, 140 mM NaCl) 
and centrifuged for 5 min at 1000 g at 4°C. The resulting cell pellets were washed 
twice in TBS and shock frozen in liquid nitrogen. 
 
Isolation of EGFP from protein extracts and reduction-alkylation. EGFP was 
purified using the µMACS
TM
 Epitope Tag Protein Isolation Kit (Miltenyi Biotec, 
Gladbach, Germany) according to the manufacturer’s protocol. Briefly, pellets from 
EGFP expressing cells treated with Aha or Hpg were resuspended in 1 ml ice cold lysis 
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X100, EDTA-free protease 
inhibitor cocktail (Complete, EDTA-free, Roche Diagnostics GmbH, Mannheim, 
Germany)) and gently shaken for 1 h at 4°C. Lysates were centrifuged at 20,000 g and for 
20 min at 4°C. The remaining supernatants were transferred to fresh tubes. Anti GFP-Tag 
MicroBeads (50 µL) were added to 600 µL of each of the respective supernatants, mixed 
and incubated for 1 h on ice. The lysate-MicroBead mixtures were applied to pre-
equilibrated µColumns in the magnetic field of the µMACS Separator, the flow-through 
was collected, and the bound EGFP was thoroughly washed four times with 200 µL wash 
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 
0.1% SDS), and once with 20 mM Tris-HCl, pH 7.5. For elution of EGFP, the columns 
were pre-incubated with 20 µL hot (95°C) elution buffer (50 mM Tris-HCl, pH 6.8, 50 
mM DTT, 1% SDS) and eluted with 50 µL of elution buffer. The isolation procedure was 
repeated twice and the eluates were combined. Following elution a buffer exchange (1x 
PBS, pH 7.8, 0.1% Triton X100, 0.1% SDS, EDTA-free protease inhibitor cocktail) was 
 S13 
performed using Zeba
TM
 Desalt Spin Columns (PIERCE, Rockford, IL, USA) according 
to the manufacturer’s protocol. Following intensive washing, 0.3 mL immobilized TCEP-
Disulfide Reducing Gel (Pierce, Rockford, IL, USA) was added to 1 mL of protein 
sample and incubated for 1 h under agitation at room temperature. Freshly prepared 
iodoacetamide at a final concentration of 10 µM was added, and the sample was 
incubated for another 30 min under agitation in the dark at room temperature. The 
procedure was finished by precipitating the TCEP resin at 1000 x g for 5 min and 
subsequent collection and desalting of the supernatant, using Zeba
TM
 Desalt Spin 
Columns (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. 
 
Verification of reaction of 12 with Hpg residues in model protein 7-Hpg-GFP. To 
examine the mass shift associated with conjugation and cleavage of 12, we compared 
MS/MS spectra of tryptic fragments of 7-Met-GFP and 7-Hpg-GFP. Proteins were 
digested with trypsin to hydrolyze peptide bonds C-terminal to lysine and arginine 
(except when followed by proline). The sequence of the protein, which contains 7 
methionine positions, is shown below. Tryptic peptides observable by mass spectrometry 
are highlighted in yellow. Methionine positions showing mass modifications are 
highlighted in green.  
 
Shown below are representative mass spectra assigned to peptide AEVKFEGMTIVNR 
(residues 62 - 74) which was observed in spectra derived from both 7-Met-GFP and 7-
Hpg-GFP. The b and y ions are shown in red and blue, respectively. For 7-Met-GFP, 
oxidation of methionine adds 16 Da to the mass of methionine. 
 S14 
 
 
 
 
 
 
For 7-Hpg-GFP, the observed mass shift is the result of two mass changes. Replacement 
of methionine with Hpg reduces the mass of the residue by 22 Da. Addition of probe 12 
in cleaved form adds a mass of 143 Da. The net mass change with respect to unmodified 
methionine is 121 Da. 
 S15 
18
12
Click chemistry
19c
10% HCO2H
20c
(CH2)6
N N
N
O
Si
R2
biotin
PhPh
(CH2)6
N N
N
OH
N3
(CH2)6
O
Si
R2 biotin
Ph
Ph
O
O
HO
N3
Chemical Formula: C6H13N3O
Exact Mass: 143.11
OH
O
NH2
S
OH
O
NH2
Chemical Formula: C5H11NO2S
Exact Mass: 149.05
Chemical Formula: C6H9NO2
Exact Mass: 127.06
difference in molecular weight for
amino acid replacement: 22 Da
mass tag on the labeled protein after cleavage: 143 Da
Total mass shift in this experiment: 143-22 = 121 Da  
 
The expected mass shift of 121 Da is observed in spectra of tryptic fragments derived 
from 7-Hpg-GFP.  
 
7-Hpg-GFP 
 
 
 
 S16 
Xcalibur software was used to set up the Orbitrap for collision-induced dissociation. The 
instrument was set to positive polarity, with a scan range of 200-1200 Da.  Mascot 
generic files (MGF) files were analyzed Sorcerer and Scaffold programs. Please refer to 
methods section for sample preparation details. 
 
 
Reference: 
1. Tsien, R. Y. The Green Fluorescent Protein Annu. Rev. Biochem. 1998, 67, 509-544. 


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Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition

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