- 1 - Enrichment of Low-molecular

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Enrichment of Low-molecular-weight Thiol Compounds
- Thiol-affinity Enrichment using Thio-tagTM MG-bead Ver. 2 (2016/2)
1. Introduction
Thiol groups in biomolecules such as glutathione and cystein-containing proteins are sensitive to redox
conditions and can function as redox switches, which turn intracellular signal transduction on or off resulting
responses to various extracellular stimuli or stresses. Although many methods have been developed for the
detection of thiol groups, few are amenable to use in the high-throughput enrichment of intact
thiol-containing molecules in small volumes or in low weights of samples. In this situation, Prof. Koike's
group (Hiroshima University) reported that a mononuclear zinc(II) complex of a macrocyclic tetraamine
(1,4,7,10-tetraazacyclododecane: cyclen) acts as a selective thiol-binding tag molecule (called “Thio-tagTM”)
in an aqueous solution. Here, we introduce a simple and efficient protocol to enrich low-molecular-weight
thiol compounds using a Thio-tagTM product under near-physiological conditions. This method is based on
magnetic-bead technology using the thiol-binding tag molecule linked to a hydrophilic cross-linked agarose
coating on a magnetic core particle (Thio-tagTM MG-bead).
2. Description of Thio-tagTM MG-bead
Thio-tagTM MG-bead provides an efficient procedure for separation of low-molecular-weight thiol
compounds from biological samples at physiological pH. The thiol enrichment procedure needs an
appropriate magnet unit and buffers for the binding, washing, and elution processes. The
commercially available product is supplied in 2-propanol. Thio-tagTM MG-bead has no irritant effect on
the skin. Stored the beads in a refrigerator at ca. 4˚C. Under the condition, the product (Zn2+-bound
form) is stable for at least 1 year.
3. Warning and Limitations
Thio-tagTM MG-bead is not for use in human diagnostic and therapeutic procedures. Do not use internally
or externally in human or animals. It's used only for research. Care should be taken to avoid contact with
the Thio-tagTM product. In the case of contact with skin or eyes, the beads should be immediately washed
off with water.
4. Advantages of Thio-tagTM MG-bead Method
# The total time for the thiol-affinity enrichment is less than 30 min.
# The method requires no expensive apparatus and the procedure is simple.
# Thio-tagTM MG-bead preferentially captures a thiol molecule (R-SH) by formation of a coordination bond
–
between thiolate (R-S ) and zinc(II) ion at pH 7.8.
# The thiolate anion bound to Thio-tagTM molecule is air-stable for a few days at room temperature.
The eluted thiol compound in an acidic fraction (e.g., 0.1 M HCl) is stable under air condition.
# Oxidized thiol derivatives such as disulfide, sulfate, and S-nitroso compounds can all be eliminated
in the flow-through and washing fractions.
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5. Basic Protocol for Separation of Thiol-containing Molecules.
Thiole-affinity separation:
2+
1) Thio-tagTM MG-beads (5 ~ 50 µL: Zn -bound form)
are placed in a 1.5-mL microtube.
2) The beads amassed by using a magnetic stand
and the storage solution is removed.
3) The beads are resuspended in 0.10 M HEPES–
NaOH buffer containing 0.10 M CH3COONa (pH
7.8, 50 ~ 200 µL) and mixed on a benchtop shaker
for 30 s, and then the liquid is removed.
This washing operation is repeated twice.
4) A sample solution consisting of 0.10 M HEPES–NaOH buffer (pH 7.8) containing thiol-containing
molecules (50 ~ 200 µL) is added in the microtube. The mixture is incubated with shaking for 2 min at
room temperature.
5) The beads are amassed magnetically and the liquid is removed as the flow-through fraction (FT).
6) The beads are resuspended in 0.10 M HEPES–NaOH buffer containing 0.10 M CH3COONa (pH 7.8,
50 ~ 200 µL) and slurry is shaken for 30 s. The beads are amassed magnetically and the cleaning
solution is isolated. This washing operation is repeated three times and the liquids are collected as
washing fractions (W1 – W3).
7) The beads are resuspended in dist. water (50 ~ 200 µL) and mixed on a benchtop shaker for 30 s,
and the liquid is isolated as the final washing fraction (W4).
8) To elute thiol-containing molecules bound to Thio-tagTM MG-beads, an acidic solution such as 0.10 M
HCl or 1%(w/v) CF3COOH (50 ~ 100 µL) is added. The slurry is incubated with shaking for 30 s. This
operation is repeated several times to give the respective elution fractions (e.g., E1 – E3).
Note: As for cystein-contining peptides, the sample in the E1 fraction was desalted using a C18-tip
prior to MS analysis. Other elution buffers: 0.10 M EDTA–NaOH (pH 7.0) for zinc(II) removal method;
10 mM dithiothreitol in 0.10 M HEPES–NaOH (pH 7.4) for thiol exchange method. Please refer to the
original article, H. Fujioka et al., J. Sep. Sci, 37, 1601-1609 (2014).
Reactivation of used Thio-tagTM MG-bead:
1) Used Thio-tagTM MG-beads are treated with 10 times volume of aqueous 0.10 M EDTA–NaOH (pH
7.0) with gentle shaking for 1 h at room temperature.
2) The beads amassed by using a magnetic stand and the liquid is removed. The beads are washed with
dist. water (e.g., mixing on a benchtop shaker) and the liquid is removed.
3) The beads are resuspended in 200 µL of 0.10 M Bis-tris–AcOH buffer containing 10 mM Zn(AcO)2 (pH
6.8) and mixed gently on a benchtop shaker for 30 min, and then the liquid is removed.
4) The beads are resuspended in 0.10 M HEPES–NaOH buffer containing 0.10 M CH3COONa (pH 7.8,
200 µL) and mixed on a benchtop shaker for 1 min, and then the liquid is removed. This washing
2+
operation is repeated twice. The Thio-tagTM beads in Zn -bound form can be stored in 2-propanol at
4˚C until next use.
Note: Thio-tagTM MG-beads used for a complex sample (e.g., cell lysate) should be discarded.
-2-
6. Solutions for Thiol-affinity Enrichment
Sol. A: Preservation solution
20%(v/v) 2-propanol/water
# 2-propanol
# distilled water for preparation of the 100 mL solution
(100 mL)
20 mL
a proper quantity
Sol. B: Binding buffer
0.10 mol/L HEPES–NaOH
# HEPES (MW = 238, 10 mmol)
# aqueous 10 M NaOH solution (ρ = 1.33 g/mL)
# distilled water for preparation of the 100 mL solution
(pH 7.8, 100 mL)
2.38 g
0.64 mL (0.85 g)
a proper quantity
Note: Check the pH of 7.8 ± 0.1 by using a pH meter.
A 10x buffer solution can be used as a 10 mL solution containing the same solutes.
Sol. C: Washing buffer
0.10 mol/L HEPES–NaOH + 0.10 mol/L CH3COONa
# HEPES (MW = 238, 10 mmol)
# CH3COONa (FW = 82, 10 mmol)
# aqueous 10 M NaOH solution (ρ = 1.33 g/mL)
# distilled water for preparation of the 100 mL solution
(pH 7.8, 100 mL)
2.38 g
0.82 g
0.64 mL (0.85 g)
a proper quantity
Note: Check the pH of 7.8 ± 0.1 by using a pH meter.
Sol. D: Elution buffer 1
# aqueous 0.10 M HCl
(pH <2, 100 mL)
Note: Another acidic aqueous solution, 1% (w/v) CF3COOH can be used similarly.
Sol. E: Elution buffer 2 (= Reactivation buffer 1)
0.10 M EDTA–NaOH buffer
# aqueous 0.10 M EDTA·2Na solution (a commercial product)
# aqueous 10 M NaOH solution (a commercial product)
for adjustment of pH to 7.0 ± 0.1 by using a pH meter.
Sol. F: Reactivation buffer 2
0.10 mol/L Bis-tris–CH3COOH + 10 mmol/L Zn(CH3COO)2
# Bis-tris (MW = 209, 10 mmol)
# aqueous 0.10 mol/L Zn(CH3COO)2 solution
# distilled water
# aqueous 1.0 M CH3COOH solution for adjustment of pH
to 6.8 ± 0.1 by using a pH meter.
# distilled water for preparation of the 100 mL solution
-3-
(pH 7.0, ca.100 mL)
100 mL
a proper quantity
(pH 6.8, 100 mL)
2.1 g
10 mL
70 mL
a proper quantity
a proper quantity
7. Selective Separation of Reduced Form of Glutathione
· Sample solution:
50 nmol of SH-glutathione, 50 nmol of SS-glutathione, and 50 nmol of
SNO-glutathione in 100 µL of Sol. C
· Washing solution: Sol. C (100 µL x 3: W1, W2, and W3); distilled water (100 µL: W4)
· Elution solution:
Sol. D (100 µL x 3: E1, E2, and E3)
The separation experiment was conducted using 50 µL of Thio-tagTM MG-beads according to the basic
protocol at room temperature except using Sol. C for preparation of the sample solution. The recoveries of
the reduced (SH-form) and oxidized (SS-form and SNO-form) species are shown in Fig. 1. The quantity in
the each fraction was analyzed by reversed phase HPLC. Total recovery of reduced glutathione was 80% in
the elution fractions. The oxidized glutathione were all eliminated in the FT and W1–W3 fractions.
Fig. 1. Recoveries of reduced and oxidized species in the each fraction.
8. Selective Separation of Cysteine-containing Peptide
· Sample solution: Tryptic digest of 5 nmol β-casein
+ 2.5 nmol oxytoceine in 100 µL of pH 7.8 HEPES buffer
Oxytoceine was prepared by reduction of oxytocin using 50 nmol TCEP in the sample solution
for 5 min at room temperature.
· Washing solution: Sol. C (100 µL x 3: W1, W2, and W3); distilled water (100 µL: W4)
· Elution solution:
Sol. D (100 µL x 2: E1 and E2)
The separation experiment was conducted using 50 µL of Thio-tagTM MG-beads according to the basic
protocol at room temperature. The separation result was analyzed by a reverse phase HPLC with a
gradient mode. The cysteine-containing peptide (reduced form of oxytocin) was preferentially eluted in the
E1 fraction.
-4-
Oxytocin: Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
(SS-form)
TCEP: tris(2-carboxyethyl)phosphine
Oxytoceine: Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SH/SH form)
Fig. 2. Chromatographic result for a mixture of tryptic digest of β-casein and oxytoceine.
E1 is the first elution fraction.
TM
References on Thio-tag Chemistry:
・Simple enrichment of thiol-containing biomolecules by using zinc(II)–cyclen-functionalized magnetic beads,
Journal of Separation Scienece, 37, 1601-1609 (2014), H. Fujioka, M. Tsunehiro, M. Kawaguchi, Y. Kuramoto,
H. Kurosaki, Y. Hieda, E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
・Role of Zinc(II) in β-Lactamase II: A Model Study with a Zinc(II)-Macrocyclic Tetraamine
(1,4,7,10-Tetraazacyclododecane, Cyclen) Complex,
Journal of American Chemical Society, 116, 8443-8449 (1994), T. Koike, M. Takamura, and E. Kimura
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