15N Metabolic Labeling Quantification Workflow in Arabidopsis Using Protein Prospector
Ruben Shrestha, Andres V. Reyes, Peter R. Baker, Zhiyong Wang, Robert J. Chalkley, Shou‐Ling Xu
Abstract
Metabolic labeling using stable isotopes is widely used for the relative quantification of proteins in proteomic studies. In plants, metabolic labeling using 15 N has great potential, but the associated complexity of data analysis has limited its usage. Here, we present the 15 N stable-isotope labeled protein quantification workflow utilizing open-access web-based software Protein Prospector. Further, we discuss several important features of 15 N labeling required to make reliable and precise protein quantification. These features include ratio adjustment based on labeling efficiency, median and interquartile range for protein ratios, isotope cluster pattern matching to flag incorrect monoisotopic peak assignment, and caching of quantification results for fast retrieval.