Phosphate-containing Metabolite Enrichment with TiO2Micro-tips

Hyun-Ju Yoo, Kristina Hakansson
2012 Bulletin of the Korean Chemical Society (Print)  
Metabolites are essential to living cells. Metabolite levels represent integrative information of cellular function, and define the phenotype of a cell or tissue in response to genetic or environmental changes. mRNA levels do not always correlate with protein levels, and translated protein may or may not be enzymatically active. Therefore, changes observed in the transcriptome or proteome do not always correspond to phenotypic alterations. Thus measurement of the metabolites synthesized by a
more » ... logical system is very important to assess genetic function and also aids the understanding of the proteome. 1 Metabolomics is the study of the full arsenal of endogenous small molecules in biological systems, with the ultimate goal of identifying biomarkers and enzymatic pathways related to human disease. The size of the metabolome is extraordinary. 2 In addition, metabolites have wide variations in chemical (e.g., molecular weight, polarity, acidity) and physical (e.g., volatility) properties, which make metabolome analysis highly challenging. Thus, targeted metabolite analysis would be valuable to selectively detect a few members of compound classes within a cell or tissue. Enrichment of specific subsets of small molecules from biological systems would greatly benefit targeted approaches. Mass spectrometry coupled with liquid chromatography (LC/MS) is preferred in metabolomics, due to the reduction of sample complexity and additional information for metabolite identification. However, polar metabolites are poorly retained with commonly used C18 columns. Incomplete separation of these metabolites may cause ion suppression even in LC/MS due to co-elution. Thus, metabolite enrichment can be beneficial to metabolomics due to the removal of co-eluted unwanted analytes. Recently, chemoselective probes based on functional groups have been used to enrich amine, acid, aldehyde, and thiol-containing compounds among polar metabolites. 3,4 However, this approach alters metabolite structure as well as tandem mass spectra. Thus, current metabolite databases cannot be used. TiO 2 has been applied to enrich phosphopeptides from complex biological matrices. 5,6 To the best of our knowledge, this is the first study in which TiO 2 was used to enrich phosphate-containing metabolites. Phosphate-containing metabolites are important in life activities such as carbon metabolism and energy conversion. Here, we present enrichment of phosphate-containing metabolites with TiO 2 microtips. Standard metabolite mixture including 7 phosphate-containing metabolites and 8 other polar metabolites was used to explore signal enhancement after using TiO2 tips. Concentrations of non phosphate-containing metabolites are much higher (~2-100 times) than those of phosphate-containing metabolites to test signal enhancement. Phosphate- Figure 1 . Mass spectra of standard metabolite mixture (Table S1 ) before (a) and after enrichment (b). This mixture contains glycerol-3-phosphate (G3P), 3-phosphoglyceric acid (3PG), glucose-6phosphate (G6P), fructose-1,6-bisphosphate (F1,6BP), ADP, ATP, and NADH. "× 10" indicates that y-axis is zoomed in by 10 times. Standard mixture composition is shown in Table S1 .
doi:10.5012/bkcs.2012.33.8.2475 fatcat:cwc6jrfjvfcelfrtlf7bskas4e