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.

ABSTRACT

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 12C-/13-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.