NanoLC MALDI MS/MS based quantitative metabolomics reveals the alteration of membrane biogenesis in oral cancer

Abstract

Cancer cells use aberrant metabolic process for proliferation and metastasis. High-throughput separation and quantification of metabolites from bio-samples is crucial in this aspect. Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), a prime technique used for metabolite identifications, suffers from several limitations in quantification studies. In this study, we have established a novel approach using conventional nanoLC-MALDI MS/MS interface for separation, identification and label free quantifications of the metabolites from biopsies. Quantification of metabolite using MALDI requires a homogeneous distribution of the matrix mixed samples on target plate for proportional time-of-flight (ToF) with the concentration of a particular metabolite present in that sample under same laser intensity. Here, crude metabolites extracted from cancer biopsies are separated and eluted as ‘fraction spots’ on MALDI target plate using nanoLC. Introducing a novel parameter, %aAUC (percentage of average area under the curve), comparative quantification of separated metabolites isolated from normal, pre-cancer and cancer biopsies has been explored. Such comparative quantification is only possible with multiple AUCs of a particular metabolite obtained from this nanoLC-MALDI MS approach. Further, selected metabolite peaks have been analyzed through MS/MS fragmentation. This approach was validated using known concentrations of an internal standard (thiourea) with its corresponding aAUC (average area under the curve). Interestingly, such comparative quantification has revealed a significant change in expression of crucial lipid metabotypes like triglyceride, phospatidyl inositol, phosphatidyl choline, glycerophospholipid, cytidine diphosphate-diacylglycerol and phosphatidylinositol bisphosphate indicating altered lipid metabolism associated membrane biogenesis in oral pre-cancer (oral submucous fibrosis) and cancer. Hence, our study successfully established an altered membrane biogenesis in oral cancer using a novel and distinct approach for separation, identification and label-free quantification of metabolites in fast growing metabotype-biomarker identification era.