CRB-SWATH: A Method for Enhancing Untargeted Precursor Ion Extraction and Automatically Constructing Their Tandem Mass Spectra from SWATH Datasets by Chromatographic Retention Behaviors

Abstract

Sequential window acquisition of all theoretical spectra (SWATH) as a typical data-independent acquisition (DIA) strategy is favorable for untargeted metabolomics. It could theoretically acquire product ions of all precursor ions, including precursor ions showing chromatographic peaks of rather poor qualities. However, existing data processing methods present limited capabilities in capturing poor-quality peaks of precursor ions. Thus, although their product ions could be acquired, their precursor ions are absent. Here, we present a new strategy, chromatographic retention behavior-SWATH (CRB-SWATH), that could unbiasedly capture poor-quality peaks and provide high resolutions of multiplexed mass spectroscopy (MS/MS) spectra in SWATH datasets. CRB-SWATH monitors CRBs of SWATH-MS signals under a series of altered elution gradients. As signals of compounds differ from noise by showing CRBs, both the precursor and fragment ions are captured, while ignoring their peak qualities. Moreover, CRB-SWATH offers good chances to resolve highly multiplexed MS/MS spectra in SWATH datasets because precursor ions coeluted in a single elution gradient often present different CRBs. In the untargeted metabolic analysis of Hela cell extracts, CRB-SWATH showed the advantage in exclusively capturing 2645 ions of poorquality peaks (i.e., tiny peaks, discontinuous ion traces, tailing peaks, zigzag peaks, etc.), accounting for 34.4% of all the untargeted precursor ions extracted. Therein, it is noteworthy that among 2116 negative ions detected in hydrophilic interaction liquid chromatography (HILIC) mode, 1284 poor-quality ion peaks (>60%) were exclusively captured by CRB-SWATH. As CRB-SWATH automatically captures a large sum of true ion peaks of poor qualities, extracts MS/MS spectra of high purities, and provides chromatographic retention behaviors of untargeted metabolites for identification and classification, it could be a useful metabolomics tool for understanding biological phenomena better.

Integrated Metabolomics and Lipidomics Approach for the Study of Metabolic Network and Early Diagnosis in Cerebral Infarction

“…The typical base peak intensity (BPI) chromatograms of the QC sample are shown in Figure S1A and B. Based on the designed serial elution gradients (G1–G7, Figure S3), the dataset of QC was deconvoluted and extracted according to the previously mentioned CRB-SWATH protocol . Finally, 12 017 and 8805 peaks were found under positive and negative modes in the CRB-SWATH strategy, respectively.…”

“…The fragmentation patterns of the mass spectra of the other metabolites were proposed (Figure S6–8). The raw files of the QC sample acquired via the CRB-SWATH approach were processed according to a previously described protocol . For targeted analysis, all scheduled MRM data were subjected to MultiQuant software (AB SCIEX, Concord, ON, Canada) and Microsoft Excel for peak integration, IS adjustment, and further data management.…”

“…The raw files of the QC sample acquired via the CRB-SWATH approach were processed according to a previously described protocol. 24 For targeted analysis, all scheduled MRM data were subjected to MultiQuant software (AB SCIEX, Concord, ON, Canada) and Microsoft Excel for peak integration, IS adjustment, and further data management. The peak area of each analyte was calculated as the ratio of the analyte area to the corresponding IS area.…”

“…As a subset of metabolomics, lipidomics shows functions similar to those of metabolomics but focuses on lipids . Up to now, the metabolic perturbation of CI has been investigated in metabolomics or lipidomics studies, wherein dysregulated metabolites and disturbed pathways were explored. ,,,− However, the reported biomarker classes were usually insufficient because there were a large number of metabolite peaks with poor quality and low intensity in the liquid chromatography-mass spectrometry (LC-MS)-based omics analysis, which were neglected in data processing. , Considering the capability of CRB-SWATH in capturing poor-quality and low-intensity peaks, it is of excellent complementarity to the typical data-independent acquisition (DIA) method for a higher metabolite coverage . Hence, the combination of routine DIA and CRB-SWATH methods has potential implications in untargeted omics studies.…”

“…22,23 Considering the capability of CRB-SWATH in capturing poor-quality and low-intensity peaks, it is of excellent complementarity to the typical data-independent acquisition (DIA) method for a higher metabolite coverage. 24 Hence, the combination of routine DIA and CRB-SWATH methods has potential implications in untargeted omics studies. Furthermore, single metabolomics or lipidomics studies cannot provide a comprehensive landscape of the molecular correlation and metabolic perturbation as both in vivo polar metabolites and lipids were associated with the pathogenesis of CI, which has been proved in previous studies.…”