Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba, UCO-CeiA3, Cordoba 14014, Spain.
Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba, UCO-CeiA3, Cordoba 14014, Spain.
J Proteomics. 2021 Feb 20;233:104082. doi: 10.1016/j.jprot.2020.104082. Epub 2021 Jan 5.
Advances in proteomic equipment, algorithms and wet protocols are being increasingly reported. Each step in the experimental workflow must be adapted and optimized to the target experimental system and objectives. The influence of the amount of peptides loaded onto the column in shotgun platforms has rarely been considered to date even though it dictates the confidence with which proteins can be identified and quantified. An experiment using variable dilutions of protein equivalent mixtures of root, leaf and seed tissue extracts of Quercus ilex was performed by subjecting BSA protein equivalent amounts of 1-100 μg to SDS-PAGE, the resulting bands being trypsin digested and peptides (10-1000 ng protein equivalents) loaded onto an LC column. Mass spectra were used to identify proteins against the in-house Q. ilex transcriptome database. Determinations included SEQUEST quantification (average of the three most abundant distinct peptides for each protein) and proteotypic peptides. The number of proteins identified was found to depend on peptide load and to peak at 2054 with 600 ng. Smaller loads led to linearly decreasing identifications from 1859 with 400 ng to 495 with 10 ng. Both quantification strategies provided similar results. The linear dynamic range was from 100 to 600 ng.
蛋白质组学设备、算法和湿实验方案的进步正在被越来越多地报道。实验工作流程的每一步都必须适应和优化目标实验系统和目标。迄今为止，尽管它决定了可以识别和定量蛋白质的置信度，但在 shotgun 平台上加载到柱子上的肽的量对其的影响很少被考虑。通过对 Quercus ilex 的根、叶和种子组织提取物的蛋白质等价混合物进行可变稀释实验，将 BSA 蛋白质等价物的量从 1 到 100μg 进行 SDS-PAGE，然后将得到的条带进行胰蛋白酶消化，并将肽（10-1000ng 蛋白质等价物）加载到 LC 柱上。质谱用于根据内部 Q. ilex 转录组数据库鉴定蛋白质。测定包括 SEQUEST 定量（每种蛋白质最丰富的三个独特肽的平均值）和蛋白质肽。鉴定的蛋白质数量被发现取决于肽的负载，在 600ng 时达到 2054 个峰值。较小的负载导致鉴定数量从 400ng 的 1859 个线性减少到 10ng 的 495 个。两种定量策略提供了相似的结果。线性动态范围为 100 到 600ng。
