Liquid chromatography-electrospray ionization-tandem mass spectrometric assay for d-aspartate N-methyltransferase activity in ark shells

Figures & data

Figure 1. The reaction catalyzed by D-aspartate N-methyltransferase.

Figure 2. ESI-MS and MS/MS spectra of FDLA derivatives of authentic N-methyl amino acids.

(a) ESI-MS spectrum of FDLA derivatives of authentic NMA. (b) ESI-MS spectrum of FDLA derivatives of authentic NMG. (c) MS/MS spectrum for the precursor ion at m/z 442 shown in a. (d) MS/MS spectrum for the precursor ion at m/z 456 shown in b.

Table 1. Validation parameters for NMDA, NMLA, NMDG, and NMLG.

	NMDA	NMLA	NMDG	NMLG
Linearity of the calibration curve ^a (r^2)	0.999	0.999	0.999	0.999
Practicality of the linear range ^b (µmol/L)	0.25–25	0.25–25	0.25–25	0.25–25
Limits of Detection ^c (fmol) (3σ, n = 5)	8.7	1.7	4.4	4.5
Limits of Quantification ^c (fmol) (10σ, n = 5)	28.9	5.7	14.6	15.0
Repeatability ^d (RSD %) (n =  6)	1.9	8.7	4.7	2.9

^aThe Linearity was evaluated by the correlation coefficient r², which was calculated by the least squares regression method.

^bMixtures of 0.01–100 μmol/L each of NMDA, NMLA, NMDG, and NMLG were measured, and the linear range showing a correlation coefficient exceeding 0.999 was determined by the least squares regression method.

^cThe limits of detection (LOD) and limits of quantification (LOQ) were calculated based on the 3σ criteria and 10σ criteria, respectively. σ: the standard deviation of each compound at 0.125 μmol/L which was half the lowest concentration (0.25μmol/L) in the linear range.

^dThe repeatability was determined as the relative standard deviation (RSD) obtained for six consecutive analyses of a mixture containing 0.250 μmol/L of each of all four enantiomers, carried out on the same day.

Figure 3. LC-ESI-MS/MS chromatograms of the FDLA derivatives of authentic N-methyl amino acids and from the assay solutions for D-aspartate N-methyltransferase in S. broughtonii.

(a) LC-ESI-MS/MS chromatograms of the FDLA derivatives of authentic NMDA and NMLA (solid line) with m/z 288 as the monitoring ion, and authentic NMDG and NMLG (dashed line) with m/z 280 as the monitoring ion. A mixture of 5 µmol/L each of NMDA, NMLA, NMDG, and NMLG was derivatized with FDLA.(b) LC-ESI-MS/MS chromatograms of FDLA derivatives in the assay solution prepared from the dialyzed tissue homogenate from the mantle of S.broughtonii, D-aspartate, and SAM without incubation at 30°C (blank). The monitoring ions used the fragment ion of m/z 288 produced from the precursor ion of m/z 442.(c) LC-ESI-MS/MS chromatograms of FDLA derivatives from the assay solution, prepared from the same dialyzed tissue homogenate used in (b), D-aspartate, and SAM with incubation for 10 min at 30°C. The monitoring ions used the fragment ion of m/z 288 produced from the precursor ion of m/z 442.

Table 2. D-aspartate N-methyltransferase activity in the tissues of S. broughtonii.

Tissue	n	LC-ESI-MS/MS method ^a	Conventional fluorescence HPLC method ^b
Mantle	3	0.582 ± 0.102	0.517 ± 0.136 ^c
Foot	3	0.025 ± 0.038	–

The activity (nmol/min per g of wet tissue) is expressed as the mean ± SD.

^aThe activity was measured using the LC-ESI-MS/MS method as described in the Materials and methods section.

^bThe activity was measured using the conventional fluorescence HPLC method as described in our previous report [14].

^cThe activity was obtained from homogenates separately prepared from another portion of the mantles from the same three clams used for the LC-ESI-MS/MS method.

Shibata K, Sugaya N, Ono W, et al. Determination of D-aspartate N-methyltransferase activity in the starfish by direct analysis of N-methyl-D-aspartate with high-performance liquid chromatography. J Chromatogr B. 2011;879:3229–3234.

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