Metabolome analysis via comprehensive two-dimensional liquid chromatography: identification of modified nucleosides from RNA metabolism

Abstract

Modified nucleosides derived from the RNA metabolism constitute an important chemical class, which are discussed as potential biomarkers in the detection of mammalian breast cancer. Not only the variability of modifications, but also the complexity of biological matrices such as urinary samples poses challenges in the analysis of modified nucleosides. In the present work, a comprehensive two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach for the analysis of modified nucleosides in biological samples was established. For prepurification of urinary samples and cell culture supernatants, we performed a cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. In order to establish a 2D-LC method, we tested numerous column combinations and chromatographic conditions. In order to determine the target compounds, we coupled the 2D-LC setup to a triple quadrupole mass spectrometer performing full scans, neutral loss scans, and multiple reaction monitoring (MRM). The combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column was found to deliver a high degree of orthogonality and adequate separation. By application of 2D-LC-MS approaches, we were able to detect 28 target compounds from RNA metabolism and crosslinked pathways in urinary samples and 26 target compounds in cell culture supernatants, respectively. This is the first demonstration of the applicability and benefit of 2D-LC-MS for the targeted metabolome analysis of modified nucleosides and compounds from crosslinked pathways in different biological matrices.

Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles

“…Phosphoribosylation, a protein modification, can also be processed by PDEs [ 131 ]. Recent innovative approaches, have utilized snake venom PDEs to digest genomic DNA into single nucleosides to study modifications of DNA [ 132 , 133 , 134 ].…”

Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines

“…Breast cancer cell lines have been investigated by application of liquid chromatography (LC)-MS [12], 2D-LC-MS [13] and nuclear magnetic resonance spectroscopy [14], before. Gas chromatography (GC) coupled to MS via electron ionization (EI) is a frequently used analytical technique for metabolite profiling [15][16][17] that allows metabolite annotation by application of mass spectral matching [18][19][20][21].…”

The volatilome – investigation of volatile organic metabolites (VOM) as potential tumor markers in patients with head and neck squamous cell carcinoma (HNSCC)

“…Conclusions about certain VOMs can be drawn by understanding the pathophysiological mechanisms, which provide insights for appropriate therapeutic treatment approaches. These markers are formed by diseased or healthy cells in the organism in response to the disease [ 37 ]. In reference to tumor diseases a change in the microbial colonization of the organism is assumed, which modulates the pattern of the VOMs.…”