Correction of Errors in Tandem Mass Spectrum Extraction Enhances Phosphopeptide Identification

The tandem mass spectrum extraction of phosphopeptides is more difficult and error-prone than that of unmodified peptides due to their lower abundance, lower ionization efficiency, the cofragmentation with other high-abundance peptides, and the use of MS3 on MS2 fragments with neutral losses. However, there are still no established methods to evaluate its correctness. Here we propose to identify and correct these errors via the combinatorial use of multiple spectrum extraction tools. We evaluated five free and two commercial extraction tools using Mascot and phosphoproteomics raw data from LTQ FT Ultra, in which RawXtract 1.9.9.2 identified the highest number of unique phosphopeptides (peptide expectation value由于磷酸化肽的丰度较低、离子化效率较低、与其他高丰度肽的共裂解以及在中性丢失的 MS2 片段上使用 MS3，因此与未修饰肽相比，磷酸化肽的串联质谱提取更为困难且易出错。然而，目前尚无建立的方法来评估其正确性。在本研究中，我们提出通过组合使用多种谱提取工具来识别和纠正这些错误。我们使用 LTQ FT Ultra 的 Mascot 和磷酸蛋白质组学原始数据评估了五种免费和两种商业提取工具，其中 RawXtract 1.9.9.2 识别的独特磷酸化肽数量最多（肽期望值）。