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.