**5. Analysis of 15N-labeled amino acid isotopomers with gas chromatography-mass spectrometry**

Unlabeled, derivatized Arg yields an ion at an *m*/*z* of 442, which corresponds to a molecular fragment containing three 14N atoms. Thus, the maximum number of 15N atoms detected is three, resulting in a mass isomer distributions of M, M+1, M+2 and M+3. These were used to calculate the isotopic enrichment in each amino acid after correction for natural isotopic contents by comparison with the mass isomer distributions measured for unlabeled standards. As shown in Fig.1, to test whether Arg is translocated from the ERM to the IRM,

**Figure 4. Labeled arginine after addition of 2 mM 13CU6 arginine to the ERM compartment for 6 weeks.** Mass isomer distributions were measured by mass spectrometry after extraction of free amino acids or hydrolysis of extracted soluble protein followed by derivatization (see methods): black bars, unlabeled arginine standard showing the natural abundance mass isomer distribution; dark grey bars, arginine extracted from unlabeled mycorrhizal root tissue; medium grey bars, arginine extracted from ERM after labeling; hatched bars, arginine extracted from mycorrhizal roots after labeling; white bars, arginine from soluble protein of mycorrhizal roots after labeling; checkered bars, arginine from soluble protein of un-colonized roots after exposure to 13CU6 arginine (positive control, showing that if arginine is available to the root tissue, it is detectable in root protein).

13CU6 arginine was added to the ERM. After 6 weeks, MS analysis of MTBSTFA-derivatized Arg isotopomer revealed that 34% of the free Arg in the ERM and 33% of the free Arg in the colonized roots showed 13CU6 labeling (M+6, Fig. 4). The mass spectra showed that the free Arg molecules in the colonized roots are either completely unlabeled (natural abundance mass isomer distribution) or labeled in all six carbon positions, thus indicating that Arg is transported intact from ERM to IRM.
