Letter reSeArCH
separate. The aqueous layer was transferred to a 4-l Erlenmeyer flask. A total of were obtained for the candidate ion. The mass of each fragment ion in the
3
l water acidified with 27 ml 1 M HCl was added to the bile extract, which caused MS/MS spectra was input into Google to search for publications that reported
a yellow-green precipitate to form. The solution was then divided into 500-ml molecules with similar fragmentation patterns. A study that contained the MS/MS
7
Nalgene bottles and centrifuged at 8,700g (7,000 r.p.m.) in an ultracentrifuge. The spectra of glutathione was found , and the glutathione spectra displayed many of
supernatant was decanted and the green, wax-like pellet was dissolved in 400 ml the fragments observed in the MS/MS spectra of the candidate ion, suggesting the
5
0% methanol. This extract was bound to a 10-g C18 solid-phase extraction (SPE) presence of similar chemical structures. Precursor ion scans in positive-ion mode
column (Waters) using a vacuum manifold (Agilent). The column was washed using m/z 179 were used to detect Cys-Gly conjugates. The data were analysed
with 50 ml 50% methanol, and then the compounds were eluted with 50 ml 100% using Analyst software.
methanol. The methanol was evaporated under compressed air to produce 600 µl
To quantify GGG in tissues, an LC–MS/MS method was developed using syn-
thetic GGG as a reference standard. GGG was detected using multiple reaction
of a concentrated extract with potent bioactivity on P2RY8-transduced cells.
HPLC purification was performed using an Agilent 1220 Infinity HPLC coupled monitoring scans with ion pair 580.3 and 179.0, and GG-Cys-Gly was detected
with an Agilent 1260 Infinity Fractionator. HPLC-grade solvents were purchased using ion pair 451.3 and 162.0. A reference standard for GG-Cys-Gly was produced
from Fisher. The maximum injection amount was 100 µl. For purifying larger by purifying supernatants from GGT5-expressing HEK293T cells incubated with
amounts of sample, the sample was injected and run multiple times per column and GGG. Three microlitres of each sample was injected into a Shimadzu Nexera X2
the corresponding fractions per minute were pooled. For each column, solvent A: HPLC, with a Synergi Polar-RP column (75 × 4.6 mm) and a mobile phase gradient
1
00% water + 0.1% formic acid; and solvent B: 100% methanol + 0.1% formic acid. consisting of A: 100% H
Fractions were collected every minute, concentrated via evaporation and tested at formic acid. 0–1 min, 50% B; 1–4 min, ramp to 80% B; 4–6 min, 80% B; 6–6.5 min,
a 1:100 dilution via bioassay. The bioactive fractions were pooled, concentrated ramp to 50% B; 6.5–8 min, 40% B. The internal standard used was LTC -d , identi-
and run on the next column. fied with ion pair 631.4/179.0. Peak area was integrated using Analyst software and
First separation: Phenomenex Luna C18, 100-Å pore size, 250 × 10.00 mm, referenced against a standard curve to calculate compound abundance.
2
O + 0.1% formic acid; and B: 100% acetonitrile + 0.1%
4
5
−1
1
0-µm particle size, part no. 00G-4094-N0. Flow rate: 2 ml min . 0–2 min, 50%
High-resolution LC–MS was performed using a Waters XEVO-G2 XS quadru-
B; 2–26.5 min, ramp to 95% B; 26.5–36.5 min, 95% B; 36.5–37 min, ramp to 50% pole time-of-flight with an Acquity UPLC equipped with a BEH C18 column. The
B; 37–38 min, 50% B.
2
mobile phase was H O with 0.05% formic acid (A) and acetonitrile with 0.05% for-
Second separation: Thermo BDS Hypersil C8, 150 × 4.6 mm, 5-µm particle size, mic acid (B). 0.1–1.9 min, 5–95% B; 1.9–2.2 min, 95% B; 2.2–2.3 min, ramp down
part no. 28205-154630. Flow rate: 1 ml min−1. 0–2 min, 50% B; 2–10 min, ramp to 5% B; 2.3–2.6 min, 5% B. Mass spectra were acquired using ESI in positive-ion
to 90% B; 10–20 min, 90% B; 20–20.5 min, ramp to 50% B; 20.5–22 min, 50% B.
mode. Metabolite databases including the human metabolome database (HMDB),
Third separation: Phenomenex Synergi Polar-RP 80-Å pore size, 150 × 4.6 mm, LipidMaps, LipidBank and Chemspider were used to search for the identity of the
4
-µm particle size, part no. 00F-4336-E0. Flow rate: 1 ml min−1. 0–4 min, 50% m/z 580.3435 candidate ion, although each query led to 0 matches.
B; 4–12 min; ramp to 95% B; 12–23 min, 95% B; 23–23.5 min, ramp to 50% B; Chemical synthesis. Unless otherwise noted, all materials used in chemical syn-
2
3.5–25 min, 50% B.
Fourth separation: Thermo APS-2 Hypersil, 150 × 4.6 mm, 5-µm particle size, Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker Avance
part no. 30705-154630. Flow rate: 1 ml min−1. 0–4 min, 50% B; 4–12 min, ramp III HD 400 MHz spectrometer.
to 95% B; 12–23 min, 95% B, 23–23.5 min, ramp to 50% B; 23.5–25 min, 50% B.
thesis were obtained commercially from MilliporeSigma and were reagent grade.
24
For geranylgeranyl bromide, the previously outlined procedure was followed.
Purification of P2RY8 ligand from cell-culture supernatants. Hepa 1-6 cells Triphenylphosphine (21.2 mg, 80.8 µmol, 1.3 eq.) was added to a solution of gera-
were grown in 16 T175 flasks using DMEM containing 10% FBS, 10 mM HEPES, nylgeraniol (20 mg, 68.9 µmol, 1 eq.) in 1 ml dry dichloromethane (DCM), stirring
2
mM glutamine and 50 IU penicillin/streptomycin. When cells were confluent, at room temperature under an atmosphere of argon. Carbon tetrabromide (29 6 mg,
the medium was replaced with RPMI containing 0.5% fatty acid-free BSA, 10 mM 89.3 µmol, 1.3 eq.) was then added, and the reaction was stirred at room tempera-
HEPES, 50 IU penicillin/streptomycin and 50 µM nicardipine, a drug that we ture for 4 h. The reaction mixture was concentrated under reduced pressure and
found increased bioactivity production specifically in Hepa 1-6 cells. To half of a small volume of n-hexane was added. The resulting precipitate was removed
the flasks, 10 µM mevastatin was also added to inhibit bioactivity production. by filtration and the filtrate was concentrated again under reduced pressure. The
After 24 h, the supernatant from each condition was collected from the cells and unstable product was used in the next step without further purification.
centrifuged at 800g (2,000 r.p.m.) to remove cell debris. Methanol was added to
For GGG, a modified version of the previously described procedure25 was used.
form a 20% methanol solution, and the solution was acidified to pH 3.5 using 1 M ꢀ-glutathione (23.0 mg, 74.9 µmol, 1.1 eq.) was dissolved in 0.5 ml 2 M NaOH,
HCl. For each condition, the solution was bound to a 10-g C18 SPE column using and approximately 1 ml ethanol was added drop-wise until the solution started to
a vacuum manifold. The columns were washed with 50 ml 50% methanol, and the become cloudy. Geranylgeranyl bromide (24.0 mg, 68.7 µmol, 1 eq.) was added
compounds were eluted with 50 ml 100% methanol and then evaporated to pro- drop-wise, and the reaction was stirred at room temperature overnight. The pH
duce 600 µl of a concentrated extract. The extracts from statin-treated and control was then adjusted to 2 by addition of 1 M HCl and the mixture was cooled in an
Hepa 1-6 cells were then simultaneously purified using the same HPLC and sol- ice bath for 20 min. The resulting precipitate was collected by filtration, washed
vent system as for bile, using the Thermo BDS Hypersil C8, Phenomenex Synergi with ice-cold ethanol and water and dried to yield geranylgeranyl glutathione as
Polar-RP and Thermo APS-2 Hypersil columns. Fractions were collected every an off-white solid (6.0 mg, 10.4 µmol, 15%).
1
minute, concentrated via evaporation and tested at a 1:100 dilution via bioassay.
6
H NMR (δ p.p.m., DMSO-d ): 8.65 (1H, app. s, NH); 8.36 (1H, d, J = 8.17 Hz,
The bioactive fractions for the control Hepa 1-6 cells were pooled, concentrated NH); 5.20–5.14 (1H, m, CH); 5.12–5.04 (3H, m, CH); 4.48–4.39 (1H, m); 3.70 (3H,
and run on the next column. The corresponding fractions from the statin-treated m); 3.32 (1H, m); 3.15 (3H, m); 2.89–2.80 (1H, m); 2.61–2.52 (1H, m); 2.42–2.22
Hepa 1-6 cells were also pooled, concentrated and run simultaneously.
SPE. C18 SPE columns were purchased from Waters (Sep-Pak, 10 g 35 cc and 1.56 (9H, s, 3 × CH3).
00 mg 6 cc versions). The SPE columns were attached to a vacuum manifold ESI high-resolution MS (m/z): Calculated for chemical formula C30
Vac Elut 20, Agilent) and pressure was maintained at 13.8–34.5 kPa (2–5 psi). The [M + H] , 580.3415; found 580.3435.
(2H, m, CH); 2.10–1.90 (12H, m, 6 × CH2); 1.64 (3H, s, CH3); 1.63 (3H, s, CH3);
5
(
49 3 6
H N O S
+
column was washed with two column-volumes of methanol, then two volumes of Crude tissue-extract preparation. Crude tissue extracts were prepared by grind-
water. Samples were typically diluted to a methanol content of 20% or lower, acid- ing mouse tissues into water (1:10 w/v), then diluting this lysate with four volumes
ified to pH 3.5 and loaded onto the column at a drop rate of 1–2 drops per second. of methanol. The mixture was centrifuged twice at 4,000g for 5 min to remove
The column was washed with 2 volumes of water and 1 volume of 50% methanol. precipitate. The supernatant was evaporated and the residue was dissolved in a
The compounds were then eluted with 1 volume of 100% methanol into glass test small amount of 100% methanol. Raw mouse bile was collected directly from the
tubes. The solution was dried down using a Thermo Reacti-Vap apparatus under gallbladder using a syringe.
compressed air and the residue was dissolved in a small amount of 100% methanol.
To obtain C18 solid-phase extracts of spleen, lymph nodes and tonsil, tissue was
Mass spectrometry. An AB SCIEX QTRAP 6500 mass spectrometer was used homogenized in 66% methanol using a Precellys 24-bead homogenizer, 1:10 w/v. For
to obtain full mass spectra (Q1), MS/MS fragment ion spectra and precursor mass spectrometry analysis, 20–100 mg of tissue was homogenized along with 15 µl
4 5
ion spectra. HPLC fractions were diluted 1:10 in HPLC-grade methanol without of a 150 nM solution of LTC -d as an internal standard. The homogenate was trans-
any additives. The diluted samples were directly injected into the ion source via ferred to a new tube. Then, 500 µl 66% methanol was used to wash the beads and was
−1
syringe at 10 µl min and ionized using electrospray ionization (ESI). Mass spectra combined with the homogenate. The mixture was centrifuged for 10 min at 4,000gin
were acquired in both positive-ion and negative-ion mode. The ion source was a microcentrifuge and the supernatant was diluted tenfold in water containing 3 mM
maintained at 100 °C, 20 CUR, 14 GS1 and 8 GS2, 5,500 IS (positive mode) or HCl. This was then bound to a 500-mg C18 SPE column, washed with 50% methanol,
−
4,500 IS (negative mode), 135 DP (positive mode) or −60 DP (negative mode), eluted with 100% methanol and concentrated down to 100 µl by evaporation.
EP 10 and CXP 10. A range of collision energy was used when performing frag- Size-exclusion centrifugal filtration of bile and cell-culture supernatant.
mentation analysis. Positive- and negative-mode fragmentation spectra (MS/MS) Amicon centrifugal filtration units with 50-kDa-cutoff membranes were
Lu, Erick
