SILARS: An Effective Stable Isotope Labeling with Ammonium Nitrate-15N in Rice Seedlings for Quantitative Proteomic Analysis  Shi-Xiang Yao, Yu Zhang, Yu-Ling Chen, Hai-Teng Deng, Jin-Yuan Liu  Molecular Plant  Volume 7, Issue 11, Pages 1697-1700 (November 2014) DOI: 10.1093/mp/ssu089 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Experimental System (A–C) and Labeling Efficiency (D, E) for SILARS in Rice Seedlings. (A) Seed germination and endosperm excision. (B) Seedlings grown in nutrient solution containing 14N (light) or 15N (heavy) ammonium nitrate as the sole nitrogen source for 21 d. No visual differences were observed between seedlings grown in the two solutions. (C) Experimental plan showing days after germination (DAG) and endosperm excision, solution changes, sucrose addition, and time of harvest. (D) Mass spectra of the [M+2H]2+ peptide GGLDFTKDDENVNSQPFMR (ribulose bisphosphate carboxylase large chain precursor, Q339G9) from LC/MS/MS analysis of a mixture of 14N(light)/15N(heavy)-labeled peptides. Seedling samples labeled with 14N or 15N for 21, 24, or 28 DAG were mixed at a 1:1 (weight:weight) ratio. The most abundant isotope peak is at the expected m/z for an isotopomer containing 26 15N atoms (indicated by the arrows). The monoisotopic [M+2H]2+ ions at m/z 1085.50 and 1098.46 correspond to the 14N-labeled peptide and 15N-labeled peptide, respectively. The m/z difference of 13.0 deconvolutes to 26.0Da, indicating the incorporation of 26 15N atoms into the peptide during SILARS. (E)15N enrichment in rice seedlings. The percent 15N enrichment was determined for SILARS-labeled seedlings at different time points and in different tissues from 28-DAG SILARS-labeled seedlings. The percent enrichment of roots was greater compared with leaves and stems. Values represent the mean ± SD from three biological replicates. Molecular Plant 2014 7, 1697-1700DOI: (10.1093/mp/ssu089) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions