Detection and analysis of protein samples of tobacco root tip tissue based on DIA LC-MS

【Objective】 The applicability of data-independent acquisition high-resolution chromatography-mass spectrometry(DIA LC-MS) for the detection of differential protein samples in root tips was analyzed to provide technical support for in-depth study of tobacco proteomics.【Method】 The root tip tissue of 6 tobacco varieties(lines) was used as the test material at 74 d after transplantation, and the cleaved protein peptides were scanned by DIA LC-MS method to analyze the applicability of this method to root-like tissue.【Result】 The total protein of root apex was extracted by TCA/acetone precipitation + SDT lysis method, and protein samples with good parallelism of horizontal bands and clear electrophoresis bands were obtained, and the sample grade evaluation all reached grade A.The constructed DDA database contained 28042 peptides, and a total of 7084 proteins were identified. The average chromatogram peak of the data was 7, the average peak capacity was 260, the main retention time(iRT) was detected, and the retention time was generally stable, the consistency score(Cscore) was 0.948 under the Q value of 0.01 standard, the median coefficient of variation (CV) was 11.5%(less than 20.0%), and the correlation coefficient was greater than 0.9. The total number of proteins identified from the root tip tissue of six tobacco varieties(lines) under DIA holographic scanning mode ranged from 4149 to 5069.【Conclusion】 The proteins identified by DIA LC-MS analysis have a wide range, a large number, high reliability, and certain reliability and stability. All quality control indicators can meet the requirements of experimental analysis, indicating that DIA LC-MS is suitable for detection and analysis of proteomic samples from tobacco root tips.