Differential analysis of amine metabolites in cells
demonstrated with dansyl or dansyl-like derivatives of
amine metabolites, using a 13C2 analog as the heavy
reagent.[14,15,22,23]
under a humidified atmosphere containing 5% CO2. The
stock suspension was diluted to a concentration of 5 × 106
cells per mL.
In this work, an isotope-labeling strategy based on an N-
hydroxysuccinimide ester reagent with a heavy reagent
incorporating six 13C atoms into a benzyl ring has been
developed. Following the evaluation of this approach using
a set of standard metabolites and cell extracts, the method
was applied to a cellular model of oxidative stress to
demonstrate its potential for differential analysis.
Differential analysis/oxidative stress experiments
Aliquots (0.4 mL) of HL60 cells (5 × 106 cells/mL) (n = 5) were
treated with either 20 μL of water (control), or 20 μL of a 20 mM
H2O2 solution (treated), for 20 min at 37°C with gentle mixing.
Metabolites were extracted (see below) and cell extracts were
evaporated to dryness. Control series were derivatized with
light and heavy reagents, whereas treated samples were
derivatized with light reagent only. Derivatized extracts were
mixed 1:1 (light/heavy), resulting in five control:control and
five treated:control samples. Quality control samples were
prepared by pooling equal aliquots of control samples.
EXPERIMENTAL
Materials
Benzoic acid, (ring-13C6)-benzoic acid, formic acid (>98%), 2-
aminoisobutyric acid (AIBA), N-acetylcysteine (NAC),
Metabolite extraction
adenosine
(Ado),
adenosine
5’-diphosphate
(ADP),
Cell culture samples were centrifuged (3500 rpm, 5 min at 4°C)
and supernatants were discarded. Pellets were then washed
twice with 1 mL cold phosphate-buffered saline (PBS). To the
resulting pellets, 300 μL MeOH/H2O 1:1 (v/v) was added
and samples were sonicated (8 min). Ice-cold MeOH (300 μL)
was then added, samples were centrifuged (14000 rpm, 8 min
at 4°C), and 550 μL of supernatant was retrieved into a new
tube. An additional 200 μL of MeOH/H2O 1:1 (v/v) was added
to the pellet, and 200 μL of resulting supernatant were
combined with the previous extract (total volume of 750 μL).
Extracts were evaporated to dryness prior to derivatization.
adenosine cyclic 2’,3’-monophosphate (AMPc), alanine (Ala),
arginine (Arg), asparagine (Asn), aspartic acid (Asp),
citrulline (Cit), cystathionine, cysteine (Cys), γ-aminobutyric acid
(GABA), glutamic acid (Glu), glutamine (Gln), glutathione
(GSH), glutathione disulfide (GSSG), glycine (Gly), histidine
(His), hydroxykynurenine (Kyn-OH), hydroxyproline (Pro-OH),
isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),
ornithine (Orn), phenylalanine (Phe), phosphorylethanolamine,
proline (Pro), serine (Ser), taurine (Tau), threonine (Thr),
tryptamine (Trpmn), tryptophan (Trp), tyrosine (Tyr) and valine
(Val) were obtained from Sigma-Aldrich (Oakville, ON, Canada).
N-Hydroxysuccinimide was purchased from Alfa Aesar (Ward
Hill, MA). N,N’-Dicyclohexylcarbodiimide, ethyl acetate and
hexane were purchased from Acros Organics-Fisher Scientific
(Ottawa, ON, Canada). Acetonitrile (ACN), monopotassium
phosphate and hydrogen peroxide (30%) were from Caledon
Laboratory Chemicals (Georgetown, ON, Canada). Methanol
was obtained from EMD (Gibbstown, NJ, USA) and ultrapure
water was supplied by a Synergy® UV purification system
(Millipore, Billerica, MA, USA). Disodium tetraborate
decahydrate was obtained from Alfa Aesar (Ward Hill, MA,
USA). Potassium chloride, sodium chloride and anhydrous
dipotassium phosphate were from Anachemia (Rouses Point,
NY, USA). The HL-60 promyelocytic human cell line was
acquired from the American Type Culture Collection
(Manassas, VA, USA). Iscove’s modified Dulbecco’s medium
and Trypan blue solution (0.4%) were from Gibco (Life
Technologies, Burlington, ON, Canada). Fetal bovine serum
(FBS) was from HyClone (Thermo Scientific, Logan, UT,
USA). Penicillin-streptomycin glutamine solution was
purchased from Wisent (St-Bruno, QC, Canada).
Relative quantitation experiments
Five extracts were created each using four aliquots of 1 mL
HL60 cell suspensions (5 × 106 cells/mL), subsequently
divided into multiple aliquots representing extracts from 1, 2
or 5 million cells each. Light and heavy labeled samples were
mixed in 1:1 proportions to prepare five series of the following
mixtures (light:heavy ratios): 1:1, 1:2, 1:5, 2:1, 2:2, 5:1, 5:5.
Derivatization reaction
Dried samples were reconstituted in 120 μL of derivatization
solvent (50 mM borate buffer containing 5% ACN, pH 9),
followed by the addition of 20 μL N-benzoyloxysuccinimide
(light or heavy, 20 mM in ACN), and incubated for 30 min
at 25°C, while mixing (Fig. 1).
LC/MS analysis
Reversed-phase liquid chromatography was performed on a
Shimadzu Nexera ultra-high-performance liquid chroma-
tography (UHPLC) system with mobile phases A (H2O) and
B (ACN), each containing 0.1% formic acid. Separation was
achieved using a Cogent C18 column (150 × 2 mm, 4 μm),
with a Phenomenex SecurityGuard C18 (4 × 2.0 mm) guard
column, at 40°C, using a gradient elution as follows: 5% B
(0–2 min), 25% (6 min); 65% (17 min); 90% (18–20 min), at a
flow rate of 0.4 mL/min.
Derivatization reagents (N-benzoyloxysuccinimide and N-
benzoyl-ring-13C6-oxy-succinimide) were synthesized in-
house (method described in the Supporting Information).
Two standard solutions of model metabolites (400 μM each)
were prepared by mixing individual stock solutions into
two standard mixes (A and B).
The LC system was coupled to a hybrid quadrupole-time-
of-flight mass spectrometer (AB Sciex Triple TOF® 5600,
Concord, ON, Canada) using a DuoSpray™ ion source
operated in positive electrospray mode. Source conditions
were: source voltage 5000 V, curtain gas 35 psi, temperature
Cell culture
HL-60 cells were cultured in Iscove’s modified Dulbecco’s
medium containing 20% (v/v) FBS, 2 mM glutamine,
100 units/mL penicillin, 100 μg/mL streptomycin, at 37°C
Rapid Commun. Mass Spectrom. 2015, 29, 1632–1640
Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm