Phenylalanine

Metabolomic profiling was performed using three separate mass spectrometry platforms run in parallel essentially as described previously (Evans et al 2009 (link)). Starting with 100 μl of plasma, small molecules were extracted in an 80 % methanol solution containing four standards (tridecanoic acid, 4-Cl-phenylalanine, 2-flurophenylglycine, and d6-cholesterol) used to monitor extraction efficiency. Clarified supernatant was split into three aliquots and dried under N₂. Additional internal standards (Standards for negative ion mode analyses included d7-glucose, d3-methionine, d3-leucine, d8-phenylalanine, d5-tryptophan, Cl-phenylalanine, Br-phenylalanine, d15-octanoic acid, d19-decanoic acid, d27-tetradecanoic acid, and d35-octadecanoic acid. Standards for positive ion mode analyses included d7-glucose, fluorophenylglycine, d3-methionine, d4-tyrosine, d3-leucine, d8-phenylalanine, d5-tryptophan, d5-hippuric acid, Cl-phenylalanine, Br-phenylalanine, d5-indole acetate, d9-progesterone, and d4-dioctylpthalate.) were added to each of three aliquots to control the quality of the chromatographic and mass spectrometric analyses. Each of the three aliquots were analyzed via a unique mass spectrometry assay: (1) gas chromatography coupled mass spectrometry (GC-MS) (2) liquid chromatography coupled mass spectrometry in positive ion mode (LC-MS pos), and (3) LC-MS in negative ion mode (LC-MS neg). For GC-MS analysis, analytes were derivatized using bistrimethyl-silyl-trifluoroacetamide and analyzed on a Trace DSQ fast-scanning single-quadruple mass spectrometer (Thermo-Finnigan). For LC-MS analyses one specimen was resuspended in 50 μl of 6.5 mM ammonium bicarbonate, pH 8, for liquid chromatography mass spectrometry (LC/MS) analysis in negative ion mode the other was resuspended in 50 μl of 0.1 % formic acid in 10 % methanol for LC/MS analysis in positive ion mode. Both resuspension buffers contained instrument internal isotopic standards used to monitor performance and serve as retention index markers. Standards for negative ion mode analyses included d7-glucose, d3-methionine, d3-leucine, d8-phenylalanine, d5-tryptophan, Cl-phenylalanine, Br-phenylalanine, d15-octanoic acid, d19-decanoic acid, d27-tetradecanoic acid, and d35-octadecanoic acid. Standards for positive ion mode analyses included d7-glucose, fluorophenylglycine, d3-methionine, d4-tyrosine, d3-leucine, d8-phenylalanine, d5-tryptophan, d5-hippuric acid, Cl-phenylalanine, Br-phenylalanine, d5-indole acetate, d9-progesterone, and d4-dioctylpthalate. Internal standards were chosen based on their broad chemical structures, biological variety and their elution spectrum on each of the arms of the platform. Chromatographic separation was completed using an ACQUITY UPLC (Waters) equipped with a Waters BEH C18 column followed by analysis with an Orbitrap Elite high resolution mass spectrometer (Thermo-Finnigan) (Evans et al 2009 (link)). For all analytic methods, metabolites were identified by matching the ion chromatographic retention index, accurate mass, and mass spectral fragmentation signatures with reference library entries created from authentic standard metabolites under the identical analytical procedure as the experimental samples (Dehaven et al 2010 (link)).

Surface staining of either erylysed blood
or splenocyte suspension (for sorting for T cells and B cells) or
lineage-depleted cells (for sorting stem/progenitor cells) was performed
by resuspending the cell pellet in ice-cold PBS containing the surface
antibody cocktail A (CD4-PeCy5 (Cat# 100410, 1:1000), CD8a-PeCy5 (Cat#
100710, 1:2000), B220-AF700 (Cat# 103231, 1:300)) for sorting B and
T cells from blood, antibody cocktail B (CD4 (Cat# 100422, 1:1000)/CD8a
(Cat# 100722, 1:1000)/B220 (Cat# 103222, 1:1000)/Ter119 (Cat# 116221,
1:500)/Gr-1 (Cat# 108416, 1:500)/CD11b (Cat# 101216, 1:1000)-all PeCy7,
c-Kit/CD117-PE (Cat# 105808, 1:1000), Sca-1-APC-Cy7 (Cat# 108126,
1:500), CD150-BV605 (Cat# 115927, 1:200), CD48-BV421 (Cat# 103428,
1:500)) for sorting stem/progenitor cells, or antibody cocktail C
(CD3-PE (Invitrogen Cat# 12-0031-83, 1:200), B220-A647 (Cat# 103226,
1:200)) for sorting B and T cells from spleen, followed by incubation
for 30 min at 4 °C in the dark. All antibodies were purchased
from BioLegend unless noted otherwise. Mast cells and macrophages
were stained with the Zombie NIRTM fixable viability dye (BioLegend,
Cat# 423105; 1:200) in 500 μL of Opti-MEM per sample for 10
min at 20 °C in the dark.
Cells were washed with 1 mL of
ice-cold PBS and centrifuged at 400g for 5 min at
4 °C, and the supernatant was then discarded. Depending on the
size of the pellet, the cells were resuspended in an appropriate volume
of ice-cold physiological water (9 g/L NaCl, usually between 500 μL
and 2 mL) containing a 1:200 ratio internal standard stock. The internal
standard stock was 6 mg/mL chloro-phenylalanine and 6 mg/mL aminoterephthalic
acid in 30% v/v 2-propanol in water. Finally, cells were filtered
through a 30 μm cell strainer (mast cells and macrophages) or
filter-cap FACS tube (all other cell types) to immediately proceed
with flow cytometry-based sorting.