Proline

Hs578T cells were seeded in equivalent amounts for 50% confluency and left it to adhere to plate. Twenty-four hours later, media was changed in one plate with SILAC heavy media (500 mL Arginine and Lysine-free media (Cambridge isotope cat. no. DMEM-500), 10% dialyzed FBS (Invitrogen cat. no. 26400-036), 1% penicillin/streptomycin antibiotics (Wisent), 50 mg Proline (Cambridge Isotopes cat. no. ULM-8333-0.1), 0.4 mM heavy Arginine (Cambridge Isotopes cat. no. CNLM-539-H-0.25), 0.275 mM heavy Lysine (Cambridge Isotopes cat. no. CNLM-291-H-0.25)) and the other with SILAC light media (500 mL Arginine and Lysine-free media (Cambridge isotope cat. no. DMEM-500), 10% dialyzed FBS (Invitrogen cat. no. 26400-036), 1% penicillin/streptomycin antibiotics (Wisent), 50 mg Proline (Cambridge Isotopes cat. no. ULM-8333-0.1), 0.4 mM Arginine (Cambridge Isotopes cat. no. ULM-8347-0.1), 0.275 mM heavy Lysine (Cambridge Isotopes cat. no. ULM-8766-0.1)). Cells were passaged for eight passages for heavy/light amino acid incorporation. The cell labeling was validated via MS to have 99.9% incorporation.

Two colonies were used for this experiment: foragers were collected and placed in experimental colonies as previously described. The sucrose solution was enriched with 1 mM of one of the two ¹³C-labeled amino acids, proline or β-alanine (Cambridge Isotope Laboratories, Tewksbury, MA, USA). Control groups were provided with sucrose solution only. Two replicates were carried out per group.
After 7 days, the hornets were killed by freezing, and the different tissues (brain, muscles, fat body, and ovaries) were dissected, collected, and dried at 60 °C for 3 days. Samples of 1 mg of each dry tissue were loaded into tin capsules. For the muscle samples, three replicates were used for each individual. For ovaries and fat body tissues, because the sample masses were insufficient for individual analysis, we pooled the tissue of several individuals (from one to seven) to reach 1 mg of dry mass. The δ¹³C (‰) values in the samples were assessed using a Picarro G2121-i Cavity Ring-Down Spectroscopy δ¹³C stable isotope analyzer with an A0502 ambient CO₂ interface, an A0201 Combustion Module, and an A0301 gas interface (CM-CRDS)^{63 (link)}. All ¹³C concentrations are expressed in δ¹³ C_VPDB.

Amino acid standards (alanine, arginine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, glutamine,
asparagine, GABA, citrulline, ornithine, taurine, tryptophan, 5-HTP,
norvaline, and kynurenine) were purchased from Sigma (St. Louis, MO).
Isotopically labeled amino acid standards (alanine (¹³C₃,¹⁵N), arginine (¹³C₆, ¹⁵N₄), aspartic acid (¹³C₄, ¹⁵N), cystine (¹³C₆, ¹⁵N₂), glutamic acid (¹³C₅, ¹⁵N), glycine (¹³C₂, ¹⁵N), histidine
(¹³C₆, ¹⁵N₃), isoleucine
(¹³C₆, ¹⁵N), leucine (¹³C₆, ¹⁵N), lysine (¹³C₆, ¹⁵N₂), methionine (¹³C₅, ¹⁵N), phenylalanine (¹³C₉, ¹⁵N), proline (¹³C₅, ¹⁵N),
serine (¹³C₃, ¹⁵N), threonine (¹³C₄, ¹⁵N), tyrosine (¹³C₉, ¹⁵N), valine (¹³C₅, ¹⁵N), citrulline (d4), GABA (¹³C₄), glutamine
(¹³C₅), asparagine(¹³C₄), ornithine (d6), tryptophan (d8), kynurenine (d4)) were obtained
from Cambridge Isotope Laboratories (Andover, MA). Stock solutions
of each compound were prepared at a concentration of 500 ng/μL
and stored at −80 °C. A 10-point calibration curve (0.01–100
pmol/μL) was prepared by serial dilutions and spiked with internal
standards at a final concentration of 5 pmol/μL. Mass spectrometry-grade
formic acid was purchased from Sigma-Aldrich (St Louis, MO), and HPLC-grade
acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ).