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，算法和湿方案的进展越来越多地被报道。实验工作流程中的每个步骤都必须根据目标实验系统和目标进行调整和优化。迄今为止，尽管它决定了可以鉴定和定量蛋白质的置信度，但在shot弹枪平台上加载到色谱柱上的肽数量的影响至今很少被考虑。通过对栎树的根，叶和种子组织提取物的蛋白质当量混合物进行可变稀释的实验，通过对BSA蛋白质当量1–100μg进行SDS-PAGE进行实验，由此产生的条带经过胰蛋白酶消化和多肽分离（10–1000 ng蛋白质当量）上样到LC色谱柱上。质谱用于鉴定内部蛋白质问：ilex转录组数据库。测定包括SEQUEST定量（每种蛋白质三个最丰富的不同肽段的平均值）和蛋白型肽段。发现鉴定出的蛋白质数量取决于肽负载量，并在2054年达到600 ng的峰值。较小的负载导致识别量从1859年的400 ng线性下降到10 ng的495。两种定量策略均提供了相似的结果。线性动态范围是100到600 ng。