Norbeck Angela D, Monroe Matthew E, Adkins Joshua N, Anderson Kevin K, Daly Don S, Smith Richard D
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
J Am Soc Mass Spectrom. 2005 Aug;16(8):1239-49. doi: 10.1016/j.jasms.2005.05.009.
The combination of mass and normalized elution time (NET) of a peptide identified by liquid chromatography-mass spectrometry (LC-MS) measurements can serve as a unique signature for that peptide. However, the specificity of an LC-MS measurement depends upon the complexity of the proteome (i.e., the number of possible peptides) and the accuracy of the LC-MS measurements. In this work, theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity. Accuracy of the LC-MS measurement of mass-NET pairs (on a 0 to 1.0 NET scale) was described by bivariate normal sampling distributions centered on the peptide signatures. Measurement accuracy (i.e., mass and NET standard deviations of +/-0.1, 1, 5, and 10 ppm, and +/-0.01 and 0.05, respectively) was varied to evaluate improvements in process quality. The spatially localized confidence score, a conditional probability of peptide uniqueness, formed the basis for the peptide identification. Application of this approach to organisms with comparatively small proteomes, such as Deinococcus radiodurans, shows that modest mass and elution time accuracies are generally adequate for confidently identifying most peptides. For more complex proteomes, more accurate measurements are required. However, the study suggests that the majority of proteins for even the human proteome should be identifiable with reasonable confidence by using LC-MS measurements with mass accuracies within +/-1 ppm and high efficiency separations having elution time measurements within +/-0.01 NET.
通过液相色谱 - 质谱（LC - MS）测量鉴定的肽段的质量与归一化洗脱时间（NET）的组合可作为该肽段的独特特征。然而，LC - MS测量的特异性取决于蛋白质组的复杂性（即可能的肽段数量）以及LC - MS测量的准确性。在这项工作中，对来自三种不同复杂程度生物体基因组的所有预测蛋白质的理论胰蛋白酶消化产物进行了特异性评估。质量 - NET对（在0到1.0的NET范围内）的LC - MS测量准确性由以肽段特征为中心的二元正态抽样分布来描述。测量准确性（即质量和NET的标准偏差分别为±0.1、1、5和10 ppm，以及±0.01和0.05）有所变化，以评估过程质量的改进。空间定位置信度得分，即肽段唯一性的条件概率，构成了肽段鉴定的基础。将这种方法应用于蛋白质组相对较小的生物体，如耐辐射球菌，结果表明适度的质量和洗脱时间准确性通常足以可靠地鉴定大多数肽段。对于更复杂的蛋白质组，则需要更精确的测量。然而，该研究表明，即使对于人类蛋白质组，通过使用质量准确性在±1 ppm以内且洗脱时间测量在±0.01 NET以内的高效分离的LC - MS测量，大多数蛋白质也应该能够以合理的置信度被鉴定出来。
