Effective Stable Isotope Labeling With Ammonium Nitrate-¹⁵N In Rice Seedlings For Quantitavie Proteomic Analysis

Quantitation of the dynamic changes of protein files in cells, tissues or organisms, is a key and long-term objective for biologists, herein to achieve this goal quantitative proteomics has emerged as a simple and robust tool. Among various quantitation strategies, metabolic labeling by stable isotope ensures different samples labeled by different isotope to be jointly processed from the very early of sample analysis, thereby minimizing sample-to-sample variations and to achieve accurate quantitation. Stable isotope labeling strategy is typically performed in Arabidopsis and tomato seedlings, because the small seed with negligible endogenous 14 N allows efficient ¹⁵ N labeling. However, perhaps due to the large seed of crops, stable isotope labeling is not yet achievable in rice and other crops.Using an ingenious strategy of endosperm excision and optimization of hydroponic nutrient solutions, stable isotope labeling with ammonium nitrate-¹⁵ N in rice seedlings（SILARS） combines hydroponic rice seedling cultivation in the case of removing the endosperm and metabolic labeling with stable isotopes using ¹⁵N-containing inorganic salts to label the entire rice seedlings. SILARS employing ¹⁵ N salts as the sole nitrogen source has achieved great success in the production of healthy-looking seedlings which contain ¹⁵ N labeled proteins with 90⁹5% incorporation during a shorter growth period of 28 days.The SILARS in combination with typical liquid chromatography electrospray ionization tandem mass spectrometry（LC-ESI-MS/MS） is suitable for quantitative rice proteomic analysis, where different samples are mixed together at the early stage of sample analysis, thus avoiding variations and errors between them, further ensures quantitation efficiency. Quantitative proteomic analysis has been performed using protein extracts of 14N/¹⁵ N mixed samples with different ratio（1:4, 1:2, 1:1, 2:1, 4:1）, protein identification and quantiation has been performed by ProLuCID software and Census software respectively, the measured ratio is very close to the known ratio, validating the good quantitation accuracy of SILARS.To illustrate the utility of this method, we have combined the higher labeling efficiency with quantitative proteomic analysis of seedlings exposed to moderate concentration of H₂O₂. In seedlings treated for 6 hours with 3.0 mM H₂O₂, a relatively mild oxidative stress, a total of 389 proteins were identified whose expression levels showed significant changes compared to untreated seedling controls. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward ROS homeostasis, cell rescue, defense, protein metabolism and carbohydrate metabolism. Stable isotope labeling provides quantitation results with relative standard deviation below 20%, validating the quantitation accuracy of SILARS.Hence, efficient labeling of entire rice seedlings by SILARS method creates new opportunities for rice quantitative proteomics to accurately determine different expression levels of proteins and facilitates to reveal important proteins functioned in rice biological processes. Furthermore, this method is also cost effective and expected to extend to quantitative proteomic studies of other cereal plants including important grain crops wheat and maize.