Introduction of 4D ‐Proteomics: A Breakthrough in Analyzing the Plant Proteomes During Stress Conditions

Proteomics is defined as the identification, quantification, and characterization of the complete set of proteins expressed in a cell or tissue under specific conditions. The last two decades have witnessed rapid advancements in proteomics technologies, including the development of the Data-Independent Acquisition (DIA) mode, which has significantly improved the sensitivity, reproducibility, and depth of proteome coverage. These advancements, together with the development of cutting-edge data analysis tools, have undoubtedly facilitated the identification of stress-responsive proteins and potential biomarkers in different organisms. However, the identification of such stress-responsive proteins, particularly in plants, remains relatively challenging because of the presence of various high-abundance proteins such as RuBisCO, which hinders the identification and subsequent characterization of these stress-responsive proteins due to their low abundance. More recently, a four-dimensional (4D) proteomics approach has been introduced, which includes "ion mobility" as the fourth dimension to classical quantitative proteomics. This 4D-proteomics method utilizes trapped ion mobility spectrometry (TIMS) combined with parallel accumulation-serial fragmentation (PASEF), which significantly enhances the sensitivity and coverage of proteomics experiments, thus allowing the detection of low-abundance proteins. This review highlights the evolution of proteomic technologies, the development of the 4D proteomics workflow, and their potential application in unraveling the molecular mechanisms underlying plant responses to environmental stress conditions. In essence, this review article provides a comprehensive overview of the state-of-the-art in proteomics, emphasizing its transformative impact on plant science research and its potential to understand crop stress resilience.