Metal Oxide-Based Selective Enrichment Combined with Stable Isotope Labeling-Mass Spectrometry Analysis for Profiling of Ribose Conjugates

Abstract

Some modified ribonucleosides in biological fluids have been evaluated as cancer-related metabolites. Detection of endogenous modified ribonucleosides in biological fluids may serve as a noninvasive cancers diagnostic method. However, determination of modified ribonucleosides is still challenging because of their low abundance and serious matrix interferences in biological fluids. Here, we developed a novel strategy for comprehensive profiling of ribose conjugates from biological fluids using metal oxide-based dispersive solid-phase extraction (DSPE) followed with in vitro stable isotope labeling and double neutral loss scan-mass spectrometry analysis (DSPE-SIL-LC-DNLS-MS). Cerium dioxide (CeO2) was used to selectively recognize and capture ribose conjugates from complex biological samples under basic environment. The enriched ribose conjugates were subsequently labeled with a pair of isotope labeling reagents (acetone and acetone-d6). The glucosidic bond of acetone labeled ribose conjugates is readily ruptured, and the generated ribose that carries an isotope tag can be lost as a neutral fragment under collision induced dissociation (CID). Since the light (acetone) and heavy (acetone-d6) labeled compounds have the same chemical structures and can generate different neutral loss fragments (NL 172 and 178 Da), it is therefore highly convenient to profile ribose conjugates by double neutral loss scan mode in mass spectrometry analysis. In this respect, the light and heavy labeled compounds were ionized at the same condition but recorded separately on MS spectra, which can significantly improve the detection specificity and facilitate the identification of ribose conjugates. Using the developed DSPE-SIL-LC-DNLS-MS strategy, we profiled the ribose conjugates in human urine, and 49 ribose conjugates were readily identified, among which 7 ribose conjugates exhibited significant contents change between healthy controls and lymphoma patients. The DSPE-SIL-LC-DNLS-MS strategy combines the selective enrichment, stable isotope labeling, and double neutral loss scan - MS analysis, which therefore can efficiently minimize false positive results, facilitate the relative quantification, and notably increase the numbers of identified ribose conjugates in biological fluids samples. Taken together, this study established a promising strategy for the effective profiling of urinary modified ribonucleosides, and simultaneous evaluation of the contents change of multiple modified ribonucleosides should provide more accurate and conclusive results for the use of urinary modified ribonucleosides as indicators of cancers.

Sensitive Determination of Onco-metabolites of D- and L-2-hydroxyglutarate Enantiomers by Chiral Derivatization Combined with Liquid Chromatography/Mass Spectrometry Analysis

“…Utilizing this highly sensitive detection method, we successfully identified and quantified D-2HG and L-2HG in human urine samples from healthy controls (n = 20) and patients with type 2 diabetes mellitus (n = 20), lung cancer (n = 20), colorectal cancer (n = 20) and nasopharyngeal carcinoma (n = 20). Before analysis, we quantified creatinine in these samples according to previously described method 23 24 . The excretion of creatinine is rather constant over a long time and it is therefore used to normalize D-2HG and L-2HG contents in different samples in the current study, which is also a standard manner in the relatively quantitative analysis of urinary metabolite 25 26 .…”

Chemical tagging for sensitive determination of uridine modifications in RNA

“…The reaction conditions, including reaction time and temperature and the concentration of CMCT, were optimized to achieve the best labelling efficiency. Aer the labelling reaction, the mixture was subjected to CeO 2 solid phase extraction (SPE) to remove excess CMCT according to a previously described procedure, 39 and the resulting derivatives were then subjected to LC-ESI-MS/MS analysis. The detailed procedure for the extraction of CMCT labelled nucleosides by CeO 2 SPE is shown in the ESI.…”

Current trends in isotope‐coded derivatization liquid chromatographic‐mass spectrometric analyses with special emphasis on their biomedical application

“…The combination of ICD, multivariate data analysis, and LC–MS/MS allowed the identification of 56 ribonucleosides in urine (Li, Jin, et al, ). The same derivatization reaction was utilized in combination with cerium dioxide‐based dispersive solid‐phase extraction and double neutral loss scan‐mass spectrometry for profiling of urinary ribonucleosides as cancer biomarkers (Chu et al, ).…”

“…For example, Higashi et al (2013) (Li, Jin, et al, 2015). The same derivatization reaction was utilized in combination with cerium dioxide-based dispersive solid-phase extraction and double neutral loss scan-mass spectrometry for profiling of urinary ribonucleosides as cancer biomarkers (Chu et al, 2015).…”