Development of Metabolomics and Lipidomics Workflows for Phosphorylated Metabolites Using Ultra-high-performance Liquid Chromatography with Tandem Mass Spectrometry

📖 The Scoop

Over the last decades, system biology and multi-omics have become an emerging field in life science. With the development of analytical instruments, especially the mass spectrometry (MS), MS based metabolomics are widely utilized to understanding the metabolic status. Due to significantly different properties of lipids, lipidomics became more and more an independent research discipline. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the most popular analytical platform in metabolomics and lipidomics. Recent progress in bioinformatical approaches has also facilitated the bottlenecking data processing steps. Although some widely used methods were established for general metabolomics and lipidomics analysis, there are still some challenging analytes like phosphorylated metabolites/lipids, which showed compromised performance. Through highly regulated phosphorylation and dephosphorylation by enzymes, phosphorylated metabolites/lipids regulate many important cellular processes. Despite their importance, the performance of their current analytical methods is not satisfying. Generally, analysis of phosphorylated analytes by LC-MS/MS face the following challenges, including low abundance, low extraction recovery, instability, regioisomers, availability of standards, etc. In order to address these issues, we developed new methods based on phosphate methylation. Accordingly, complete workflows from sample preparation to data processing were developed and validated in biological matrix. In the 1st project, a targeted LC-MS/MS method of fatty acyl-CoAs (acyl-CoAs) with different chain lengths was established. Acyl-CoAs are central metabolic intermediates in numerous biological processes. Due to diverse fatty acyl compositions, acyl-CoAs vary greatly in polarity, which poses great challenges to their chromatographic separation. Currently, short chain acyl-CoAs are separated by HILIC or acidic RP-LC, while medium-, long- and very long-chain acyl-CoAs are analysed in basic RP-LC. Additionally, carryover in LC-MS/MS instrumentation requires extra flushing/cleaning procedures. To address these problems, we developed phosphate methylation strategy for acyl-CoAs. In the sample preparation, mixed-mode SPE was optimized to be compatible with methylation. SRM transitions in MS detection was constructed with fragmentation rule for all methylated acyl-CoAs. Uniformly 13C labelled yeast extract was used as internal standards for accurate quantification. Odd chain and stable isotope labelled analogs were used as surrogate calibrators. The LOQs were between 16.9 nM and 4.2 nM. The method was applied in cultured HeLa cells and human platelets of coronary artery disease patients. The results showed that the established method can be used to profile acyl-CoAs in biological samples. In the 2nd project, an isomer selective LC-MS/MS method of PIPx was developed. PIPx is a family of low abundant phospholipids commonly found in eukaryotic cells. Starting from phosphoinositol (PI), PIPx family consists of three lipid classes (PIP, PIP2, and PIP3) according to phosphorylation degree. There are three distinct regioisomers in each class of PIP and PIP2. As phospholipids, PIPx has two fatty acyl groups at the sn1 and sn2 position of a glycerol backbone. Currently, methods capable of regioisomer separation and fatty acyl coverage are still missing. To develop a comprehensive method, we utilized polysaccharide chiral column with data independent acquisition technique. Stable isotope labelled PIPx were characterized and used as internal standards. The established method was applied to several biological samples, including NIST SRM1950 plasma, Pichia Pastoris, and cultured HeLa cells. The results indicate this method could effectively monitor PIPx profiles in real samples and facilitate our understanding of physiological and pathological conditions. In the 3rd and 4th project, improved LC-MS/MS methods of IPx and SPx were developed. Compared to PIPx and acyl-CoAs, IPx and SPx are more polar and hydrophilic. To apply the phosphate methylation strategy, a new derivatization solvent mixture was developed and optimized. To improve extraction recovery, an extraction kit compromised of TiO2 and centrifuge filter was established. Differential isotope labelling methylation was utilized to generate stable isotope labelled analogs in both projects (internal standards in IPx project, surrogate calibrators in SPx project). Cholesterol-ether bonded RP column was used to separate IPx regioisomers, especially I(1,4,5)P3. Porous graphite carbon column was used to separate SPx in glycolysis pathway. After validation in biological matrix, the methods were applied in different biological samples, including NIST SRM1950 plasma, cultured HeLa cells, and human platelets.

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