15N metabolic labeling of mammalian tissue with slow protein turnover

Abstract

We previously reported the metabolic 15N labeling of a rat where enrichment ranged from 94% to 74%. We report here an improved labeling strategy which generates 94% 15N enrichment throughout all tissues of the rat. A high 15N enrichment of the internal standard is necessary for accurate quantitation, and thus, this approach will allow quantitative mass spectrometry analysis of animal models of disease targeting any tissue.

Figure 1. Diagram of the enrichment protocols

A, In protocol 1, a female rat on pregnancy day 2 (E2) was placed on the ¹⁵N enriched diet. It remained on the ¹⁵N diet during pregnancy and the weaning of its pups. After weaning, the pups were placed on the ¹⁵N diet for 25 days. B, In protocol 2, a weaned female rat was placed on a ¹⁵N diet and remained on the ¹⁵N diet during mating, pregnancy and the weaning of its pups. After weaning, the pups were placed on the ¹⁵N diet for 25 days.

Figure 2. A, The number of pups in a litter was unaffected by the metabolic labeling of the mothers

The white bars represent litters from ¹⁵N labeled rats and the black bars represent litters from rats on the control diet. In protocol 1, one pregnant rat was placed on the ¹⁵N diet and one pregnant rat on the control diet. In protocol 2, two female rats were placed on the ¹⁵N diet and two on the control diet before mating. B, The percent ¹⁵N enrichment of liver and brain tissues from maternal rats. The black bars represent tissues from a maternal rat from protocol 1 and the white boxes represent tissues from a maternal rat from protocol 2. Values represent averages +/- SD.

Figure 3. The percent ¹⁵N enrichment of liver and brain tissues from rat pups during development

A, The percent ¹⁵N enrichment was determined for liver tissue at 3 developmental ages, p1, p20, and p45. There were significant differences (** p < .001) detected between the two enrichment protocol at p1 and p20, but no significant differences were detected at p45. B, The percent ¹⁵N enrichment was determined for brain tissue at 3 developmental ages, p1, p20, and p45. There were significant differences (** p < .001) detected between the two enrichment protocol at p1 and p20. In contrast to the liver tissue, there was a significant difference detected at p45 between the two enrichment protocols. Values represent averages +/- SD.

Figure 4. The ¹⁵N enrichment affects the isotopic distribution

The peptide, K.EITALAPSTMK.I, was identified in both p45 (A) and p1 (B) brain samples from protocol 2. The peptide was measured to be 95% enriched in the p45 sample and 68% enriched in the p1 sample. The isotopic distribution of the ¹⁴N peptide is identical in both mass spectra with the major peak at 1161.5 m/z, but the isotopic distribution for the ¹⁵N peptide is different between the two samples. The major isotopic peak of the ¹⁵N peptide in p45 is at 1173.5 m/z, while it is at 1170.5 m/z in p1. In addition, the ¹⁵N isotopic distribution is broader, less intense in p1 compared to p45. The y-axis is relative abundance and the x-axis is mass/charge.

Figure 5. The ¹⁵N enrichment of skeletal muscle

A, The percent ¹⁵N enrichment was determined for skeletal muscle from the maternal rats. Consistent with other maternal tissues, the percent enrichment of skeletal muscle was greater from the maternal rat from protocol 2 compared to protocol 1. B, The percent ¹⁵N enrichment was determined for skeletal muscle at p20, and p45. There were significant differences (** p < .001) detected between the two enrichment protocol both at p20 and p45. Values represent averages +/- SD.