Profiling of carbonyl compounds in serum by stable isotope labeling - Double precursor ion scan - Mass spectrometry analysis

- Four labeling reagents were synthesized and compared to label carbonyl compounds.
- IL-LC-DPIS-MS analytical strategy was developed for profiling and quantitation of carbonyl compounds in human serum.
- 156 carbonyl compounds candidates were detected in human serum.
- 44 carbonyl compounds exhibited significant difference between myelogenous leukemia patients and healthy controls.

Carbonyl compounds are considered as the potential biomarkers for oxidative stress and many types of diseases; therefore their determination may serve as indicator for early clinical diagnosis. Here we developed a strategy based on isotope labeling combined with liquid chromatography-double precursor ion scan-mass spectrometry (IL-LC-DPIS-MS) analysis for comprehensive profiling and relative quantitation of carbonyl compounds in human serum. First, we chose labeling reagents (2-(2-hydrazinyl-2-oxoethyl)isoquinolin-2-ium bromide, HIQB; N,N,N-triethyl-2-hydrazinyl-2-oxoethanaminium bromide, THB; Girard reagent T, GT; Girard reagent P, GP), all of which contain reactive group, isotopically labeled moiety and ionizable group to selectively label carbonyl compounds. Since HIQB labeling offered the best detection sensitivities for carbonyl compounds among these labeling reagents, we used HIQB and the corresponding isotope-labeled reagent of d7-HIQB as the optimal isotope-labeled reagent. The HIQB and d7-HIQB labeled carbonyl compounds can generate two characteristic product ions of m/z 130.1/137.1 under collision-induced dissociation (CID), which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. Using this strategy, 156 carbonyl compounds candidates were detected in human serum, 12 of which were further identified by commercial standards. Subsequently, a targeted method using multiple reaction monitoring (MRM) detection mode was developed for relative quantification of carbonyl compounds in human serum from myelogenous leukemia (ML) patients and healthy controls. As a result, 44 carbonyl compounds were found to have significant difference between ML patients and healthy controls, suggesting that these carbonyl compounds may play certain roles in ML and also can serve as indicators for ML. Taken together, the isotope labeling combined with tandem mass spectrometry analysis demonstrated to be a powerful strategy for identification and quantification of carbonyl compounds in serum samples.

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