4.2 Overview of DIA quantitative proteomics analysis
Due to the complex mechanism of rice quality formation, this study conducted the proteomics analysis on physiological pathways such as starch synthesis and metabolism, storage protein accumulation, and plant photosynthesis under warming conditions, and to identify the key regulation factors in pathways related to temperature response. In recent years, proteomics-based mass spectrometry has made significant progress from sample preparation to liquid chromatography and instrument detection, making it possible to identify more specific expressed proteins in cells or tissues with excellent accuracy and repeatability (Tsou et al., 2015). Data independent acquisition (DIA) is widely used in proteomics analysis due to its higher protein coverage rate and reliable data acquisition ability (Renaud & Sumarah, 2016, Searle et al., 2015). Compared with iTRAQ, the advantage of DIA technology is that it can effectively measure protein molecules with extremely low abundance in complex samples, which greatly improves the reliability of quantitative analysis and has high quantitative accuracy and repeatability. In this study, samples of interest went through mass spectrometry data collection in data dependent acquisition (DDA) mode. MaxQuant was then used to carry out database search identification process and obtain all detectable non-redundant high-quality MS/MS spectral information as DIA spectral library, which contains fragment ion intensity and retention time describing the peak characteristics of the peptide, for quantification. Here, we identified 23968 unique peptides and 5872 unique proteins, which could be specifically regulated by the increased temperature during rice grain filling. Those DEPs coordinate and execute their biological behaviors based on their metabolic pathways. Therefore, KEGG pathway-based analysis would be contributed to further understand their biological functions. From our results, these identified specifically expressed proteins have large differences in temporal and spatial characteristics, which provided the obstacles to our further identification and screening of key regulators. Therefore, returning to the essential relationship between grain-filling and quality formation, we further screened the key proteins that we believe are specifically regulated by warming during the quality formation process from the perspectives of plant photosynthesis, grain starch and storage protein accumulations.