De Novo Cleaning of Chimeric MS/MS Spectra for LC-MS/MS-Based Metabolomics

The purity of tandem mass spectrometry (MS/MS) is essential to MS/MS-based metabolite annotation and unknown exploration. This work presents a de novo approach to cleaning chimeric MS/MS spectra generated in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. The assumption is that true fragments and their precursors are well correlated across the samples in a study, while false or contamination fragments are rather independent. Using data simulation, this work starts with an investigation of the negative effects of chimeric MS/MS spectra on spectral similarity analysis and molecular networking. Next, the characteristics of true and false fragments in chimeric MS/MS spectra were investigated using MS/MS of chemical standards. We recognized three fragment peak attributes indicative of whether a peak is a false fragment, including (1) intensity ratio fluctuation, (2) appearance rate, and (3) relative intensity. Using these attributes, we tested three machine learning models and identified XGBoost as the best model to achieve an area under the precision-recall curve of 0.98 for a clear separation between true and false fragments. Based on the trained model, we constructed an automated bioinformatic platform, DNMS2Purifier (short for de novo MS2Purifier), for metabolic features from metabolomics studies. DNMS2Purifier recognizes and processes chimeric MS/MS spectra without additional sample analysis or library confirmation. DNMS2Purifer was evaluated on a metabolomics data set generated with different MS/MS precursor isolation windows. It successfully captured the increase in the number of false fragments from the increased isolation window. DNMS2Purifier was also compared to MS2Purifier, an existing MS/MS spectral cleaning tool based on the addition of data-independent acquisition (DIA) analysis. Results indicated that DNMS2Purifier uniquely recognizes false fragments, which complements the previous DIA-based approach. Finally, DNMS2Purifier was demonstrated using a real experimental metabolomics study, showing improved MS/MS spectral quality and leading to an improved spectral match ratio and molecular networking outcome.