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Abstract
Background: 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. Results:. 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 Significance: 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.
Supplementary materials
Title
Improved Quantification of Carbonyl Sub-metabolome by Liquid Chromatography Mass Spectrometry using a Fragment Controlled Multiplexed Isotopic Tag
Description
Synthesis approach for the 5-plex DiMe-FP-NHNH2 tags; the list and structure of model metabolites; the list and structures of urine carbonyl metabolite standards; LC-MS/MS analysis of 5-plex DiMe-FP-NHNH2 tags; 1H NMR of DiMe-FP-NHNH2 (Δ0); 13C NMR of DiMe-FP-NHNH2 (Δ0); the fragmentation behavior of the DiMe-FP-NHNH2 tag (Δ0, m/z 305.19); the labeling scheme of 12 prostate cancer study samples; examples of scoring for IP, DI, and NL. MS1 and MS2 spectra at rt 14.23 min; relative quantification of aldehyde candidate with m/z 671.35 (Δ0)); absolute quantification of aldehyde candidate with m/z 655.35 (Δ0)); the fragmentation behavior of 17 model metabolites; the MS1 isotope patterns of 5-plex DiMe-FP-estrone mixed at 1:1:1:1:1, 1:2:5:10:20 and 20:10:5:2:1; the fragmentation of dansylhydrazine and 2,4-dinitrophenylhydrazine labeled propanal at different CEs.
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