Unraveling the potential of segment scan mass spectral acquisition for chemical isotope labeling LC-MS-based metabolome analysis: Performance assessment across different types of biological samples.

Background: Chemical isotope labeling (CIL) LC-MS is a powerful tool for metabolome analysis with high metabolomic coverage and quantification accuracy. In CIL LC-MS, the overall metabolite detection efficiency using Orbitrap MS can be further improved by employing a segment scan method where the full m/z range is divided into multiple segments for spectral acquisition with a significant increase in the in-spectrum dynamic range. Considering the metabolic complexity in different types of biological samples (e.g., feces, urine, serum/plasma, cell/tissue extracts, saliva, etc.), we report the development and evaluation of the segment scan method for metabolome analysis of different sample types.

Results: It was found that sample complexity significantly influenced the performance of the segment scan method. In metabolically complex samples such as feces and urine, the method yielded a substantial increase (up to 94 %) in detected peak pairs or metabolites, compared to conventional full scan. Conversely, less complex samples like saliva exhibited more modest gains (approximately 25 %). Based on the observations, a 120-m/z segment scan method was determined as a routine approach for CIL LC-Orbitrap-MS-based metabolomics with good compatibility with different types of biological samples. For this method, a further investigation on relative quantification accuracy was done. The peak area ratios of C-/-labeled metabolites were slightly reduced with 72%-84 % of peak pairs falling within the ±25 % range of the anticipated peak ratio of 1.0 among different samples, as opposed to 81%-90 % in the full scan, which was attributed to the inclusion of more low-abundance peak pairs within the narrow MS segments. However, the overall peak ratio measurement precision was not significantly affected by the segment scan.

Significance And Novelty: The segment scan method was found to be useful for CIL LC-Orbitrap-MS-based metabolome analysis of different types of samples with significant improvement in metabolite detectability (25-94 % increase), compared to the conventional full scan method.