Improved quantification of carbonyl sub-metabolome by liquid chromatography mass spectrometry using a fragment controlled multiplexed isotopic tag

Carbonyl-containing metabolites are a class of key intermediate in metabolism, which has potentials to be biomarkers. Since their poor ionization, derivatization reagents, such as dansylhydrazine, are usually used to improve the sensitivity and/or to facilitate quantification. However, most current carbonyl derivatization reagents only have two channels, one is isotopically labeled and the other one is non-labeled. To quantify more samples in a run and using data-independent acquisition (DIA) mode to get comprehensive and unbiased mass fragmentation, we proposed a fragment-controlled isotopic tag, called DiMe-FP-NHNH2 (FP) which has five channels: Δ0, Δ3, Δ6, Δ9, and Δ12, thus up to 5 samples can be analyzed in a run. The most important improvement is that the FP tag can produce multiple characteristic signals in tandem mass, diagnostic ions and neutral losses, which helps to selectively detect aldehydes/ketones for targeted and untargeted analysis. To exhibit all capabilities of the FP tag, we mimicked an untargeted metabolomics experiment, which comprises two steps. First, discovery step, using Data-Independent Analysis (SWATH-MS) and the labeling of two channels (Δ0 and Δ3), we picked out aldehyde/ketone from the pooled urine samples based on three characteristic signals, including isotope patterns, diagnostic ions, and neutral losses. Second, five-plex quantification, relative and absolute quantification were achieved in a single LC-MS analysis. Notably, because of different nominal masses, the FP tag can be used on any low or high resolution mass spectrometers. The benefits and performance of the FP tag are demonstrated by the analysis of urine samples collected from patients from a prostate cancer study, in which more than a thousand features were found based on MS1 fingerprint, but only around 120 aldehyde/ketone candidates were confirmed with characteristic signals and nine of which were quantified showing significant differences from healthy and reference urine samples.