**6. Conclusion**

240 Chromatography – The Most Versatile Method of Chemical Analysis

nitrogens by fragmentation at 70 eV.

**chromatography-mass spectrometry** 

amino acid was determined by measuring the M-57 ions which result from the loss of a tbutyl group (i.e., [M - C(CH3)3]) from the molecules of MTBSTFA derivatives, except for Arg whose m-188 ion was used. The Arg ion examined had a *m*/*z* of 442, which corresponds to M-188 (molecular ion minus 188) fragment arising from the loss of one guanido nitrogen together with a tBDMS and DMS group from the tetra-substituted *tert*butyldimethylsilyl(tBDMS)-derivatized Arg (Fig. 2). When using [guanido-2-15N] Arg, we observed an ion at an *m*/*z* of 443 (M-189, molecular ion minus 189). This isotopomer corresponds to the derivatized [guanido-2-15N]Arg because the ion loses one of the guanido

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).

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

Chromatographic and mass spectrometry analysis of amino acids, in combination with isotopic tracing, shows that various forms of N sources (NO3- , NH4+, amino acids) are taken up, assimilated, and incorporated into Arg by the ERM. The accumulated Arg as well as polyP is then bidirectionally translocated along the coenocytic fungal hyphae from the ERM to the IRM or from the mycorrhizal compartment tissue to the ERM. Arg is catabolized through catabolic arm of the urea cycle ( utilizing arginase and urease activities) in the IRM, releasing NH3/NH4+ in the arbuscules. The NH3/NH4+ acquired by the plant is either transported into adjacent cells or immediately incorporated into AAs, as shown in Fig.1 (modified from Govindarajulul et al.[29]).

However, although Ala, Gly, Val, Leu, Ileu, Pro, Ser, Thr, Phe, Asn, Asp, Glu, Orn, Gln, Arg and tyrosine(Tyr) are detected, GC-MS of samples from AM fungal tissues performed after MTBSTFA-derivatized amino acids cleaned up on a cation exchange column (DOWEX 50 \*4- 200, hydrogen form) does not detect some of S-containing amino acids and basic amino acids lysine (Lys) and His. Some amino acids cannot be quantified with chromatographic MS due to non-linear response. Nevertheless, analysis of PITC-derivatized amino acids with HPLC shows excellent linear relationship between the molar concentrations of amino acids and peak areas in the chromatogram, and thereby can be used for effective quantification. Although many techniques are already in use in this field, we will need some novel methods, yet to be developed, to achieve a simultaneous measurement and identification of all free amino acids in biological tissues.
