or conductometric detection9,13-16 were used to circumvent
metabolites insufficient UV absorbance and, consequently, low
UV detection sensitivity.
new derivatization reagent targeting multiple functional groups
that is more suitable for quantification of specific metabolic
pathways as well as differential global metabolomics. An in vitro
aniline derivatization is used that allows for absolute quantification
of known compounds or relative quantification of unknown
compounds without requiring standards. Moreover large numbers
of analytes can be analyzed in a single LC-MS run.
The utility of in vitro aniline derivatization was applied to the
quantification of intermediates in central carbon and energy
metabolism. Among the whole cellular metabolic network, central
carbon metabolism, composed of glycolysis, the pentose phos-
phate pathway, and the tricarboxylic acid cycle (TCA), plays a
key function in substrate degradation, energy and cofactor
regeneration, and biosynthetical precursor supply. There are more
than 35 intermediates that belong to several categories of chemical
compounds: phosphorylated sugars, phosphocarboxylic acids,
carboxylic acids, nucleotides, and cofactors. Simultaneous analysis
of these compounds is a challenging analytical problem. This
paper describes a new in vitro 13C6 labeling method that allows
accurate determination of most intermediates involved in central
carbon and energy metabolism in a single 30 min reversed-phase
liquid chromatography-mass spectrometry (RPLC-MS) run.
One of the most common ways of analyzing metabolites is
through separation by gas or liquid chromatography followed by
identification and quantification through mass spectrometry
(MS).17,18 Signal intensity of an analyte in MS depends on its
concentration and ionization efficiency. Ionization efficiency not
only varies between analytes but can depend on other components
in the matrix, particularly in the case of electrospray ionization
as used in LC-MS. This problem can be circumvented in LC-MS
quantification through the use of a 13C-coded internal standard
that coelutes with the analyte and has an ionization environment
identical to the analyte. Synthesizing the requisite 13C-coded
internal standard is generally simple when the number of analytes
being determined is small. When large, the requisite number of
syntheses can become prohibitive. Although it is possible to
biosynthesize 13C-coded metabolites,19 a comprehensive collection
of internal standard metabolites is generally not available. Some
have used standard addition methods for MS quantification19-22
to circumvent this problem. However, the MS response can
change over time due to changes in the MS instrument.23
The paper reports a new postbiosynthetic (in vitro) stable
isotope encoding procedure called group specific internal standard
technology (GSIST).24,25 In GSIST, metabolites from control
samples (or metabolite standards) and experimental samples are
derivatized with chemically identical but isotopically distinct
labeling agents. In effect, sample components are chemically
coded according to their sample origin. After mixing these
derivatized metabolites, each molecule from the control or
standard sample serves as an internal standard for determining
the concentration of the corresponding compounds in the experi-
mental sample. Recent studies in our laboratory have focused on
derivatizing agents targeting primary amines10 and carboxyl
groups.24 Although these coding agents work well for specific
classes of molecules, they have some limitations in global and
pathway-targeted approaches since not all molecules contain the
same functional groups. For this reason we have introduced a
EXPERIMENTAL SECTION
Materials and Reagents. All metabolite standards, aniline,
aniline-13C6, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hy-
drochloride (EDC), tributylamine (TBA), triethylamine (TEA), and
HPLC grade water were purchased from Sigma-Aldrich (St. Louis,
MO). HPLC grade acetonitrile (ACN) was obtained from Mallinck-
rodt Baker (Phillipsburg, NJ).
Yeast Growth and Fermentation. S. cerevisiae (ATCC 4124)
was inoculated directly from the agar plates into 5 mL of YEP +
2% glucose medium. The cultures were incubated in a shaker at
30 °C and 200 rpm and grown aerobically overnight. The following
morning, the culture were transferred directly to 100 mL YEP +
2% glucose in a 300 mL Erlenmeyer flask equipped with a side
arm (Bellco), which allows for direct monitoring of the growth of
yeast cultures by a Klett colorimeter (Manostat Corp.). The
cultures were incubated as described above until cell density
reached 500 KU. At this point, 24 mL of (50%) glucose was added
to the flask. The flask was then sealed with Saran wrap to allow
fermentation to proceed under largely anaerobic conditions. The
cultures were incubated as described above, with cell growth
monitoring by Klett colorimeter. One milliliter samples of the
mixture were removed at proscribed intervals to monitor fermen-
tation. The sample for intracellular metabolite analysis was taken
3 h after fermentation started. Glucose and fermentation products
such as glycerol, acetic acid, and ethanol were analyzed by high-
performance liquid chromatography (HPLC) using HPX 87H (8
mm × 300 mm, Bio-Rad Laboratories, CA).
(12) Picioreanu, S.; Poels, I.; Frank, J.; van Dam, J. C.; van Dedem, G. W.; Nagels,
L. J. Anal. Chem. 2000, 72, 2029–2034
(13) Bhattacharya, M.; Fuhrman, L.; Ingram, A.; Nickerson, K. W.; Conway, T.
Anal. Biochem. 1995, 232, 98–106
(14) Hull, S. R.; Montgomery, R. Anal. Biochem. 1994, 222, 49–54
(15) Ritter, J. B.; Genzel, Y.; Reichl, U. J. Chromatogr., B: Anal. Technol. Biomed.
Life Sci. 2006, 843, 216–226
(16) Vogt, A. M.; Ackermann, C.; Noe, T.; Jensen, D.; Kubler, W. Biochem.
Biophys. Res. Commun. 1998, 248, 527–532
(17) Stephanopoulos, G.; Alper, H.; Moxley, J. Nat. Biotechnol. 2004, 22, 1261–
1267
.
.
.
.
.
.
(18) Wamelink, M. M.; Struys, E. A.; Huck, J. H.; Roos, B.; van der Knaap, M. S.;
Jakobs, C.; Verhoeven, N. M. J. Chromatogr., B: Anal. Technol. Biomed.
Life Sci. 2005, 823, 18–25
(19) Huck, J. H.; Struys, E. A.; Verhoeven, N. M.; Jakobs, C.; van der Knaap,
M. S. Clin. Chem. 2003, 49, 1375–1380
(20) Buchholz, A.; Takors, R.; Wandrey, C. Anal. Biochem. 2001, 295, 129–
137
(21) Luo, B.; Groenke, K.; Takors, R.; Wandrey, C.; Oldiges, M. J. Chromatogr.,
A 2007, 1147, 153–164
(22) van Dam, J. C.; Eman, M. R.; Frank, J.; Lange, H. C.; van Dedem, G. W. K.;
Heijnen, S. J. Anal. Chim. Acta 2002, 460, 209–218
(23) Coulier, L.; Bas, R.; Jaspersen, S.; Verheij, E.; van der Werf, M. J.;
Hankemeier, T. Anal. Chem. 2006, 78, 6573–6582
(24) Yang, W. C.; Adamec, J.; Regnier, F. E. Anal. Chem. 2007, 79, 5150–5157
(25) Yang, W. C.; Mirzaei, H.; Liu, X. P.; Regnier, F. E. Anal. Chem. 2006, 78,
4702–4708
.
.
Sample Preparation. Sampling was performed as described
by Gonzales et al.26 and Lange et al.27 Briefly, 5 mL of yeast culture
was sprayed into 50 mL centrifugation tubes (Oak Ridge centrifu-
gation tube, FEP) containing 26 mL of cold solution with 60%
(v/v) analytical grade methanol (Mallinckrodt), kept at -45 °C
.
.
.
.
.
(26) Gonzalez, B.; Francois, J.; Renaud, M. Yeast 1997, 13, 1347–1355
(27) Lange, H. C.; Eman, M.; van Zuijlen, G.; Visser, D.; van Dam, J. C.; Frank,
J.; de Mattos, M. J.; Heijnen, J. J. Biotechnol. Bioeng. 2001, 75, 406–415
.
.
.
Analytical Chemistry, Vol. 80, No. 24, December 15, 2008 9509