Stable Isotope Tracing Experiments Using LC-MS.

Metabolomics has emerged as a pivotal field in understanding cellular function, particularly in the context of disease. In numerous diseases, including cancer, alterations in metabolism play an essential role in disease progression and drug response. Hence, unraveling the metabolic rewiring is of importance to find novel diagnostic and therapeutic strategies. Isotope tracing is a powerful technique for delving deeper into the metabolic wiring of cells. By tracking an isotopically labeled substrate through biochemical reactions in the cell, this technique provides a dynamic understanding of cellular metabolism. This chapter outlines a robust isotope tracing protocol utilizing high-resolution mass spectrometry coupled to liquid chromatography in cell culture-based models. We cover essential aspects of experimental design and analyses, providing a valuable resource for researchers aiming to employ isotopic tracing.

Targeting coenzyme Q10 synthesis overcomes bortezomib resistance in multiple myeloma.

During the development of drug resistance, multiple myeloma (MM) cells undergo changes to their metabolism. However, how these metabolic changes can be exploited to improve treatment efficacy is not known. Here we demonstrate that targeting coenzyme Q10 (CoQ) biosynthesis through the mevalonate pathway works in synergy with the proteasome inhibitor bortezomib (BTZ) in MM. We show that gene expression signatures relating to the mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC) predispose to clinical BTZ resistance and poor prognosis in MM patients. Mechanistically, BTZ-resistant cells show increased activity of glutamine-driven TCA cycle and oxidative phosphorylation, together with an increased vulnerability towards ETC inhibition. Moreover, BTZ resistance is accompanied by high levels of the mitochondrial electron carrier CoQ, while the mevalonate pathway inhibitor simvastatin increases cell death and decreases CoQ levels, specifically in BTZ-resistant cells. Both in vitro and in vivo, simvastatin enhances the effect of bortezomib treatment. Our study links CoQ synthesis to drug resistance in MM and provides a novel avenue for improving BTZ responses through statin-induced inhibition of mitochondrial metabolism.

Distinct lipid compositions of two types of human prostasomes.

Prostasomes are vesicles secreted by prostate epithelial cells and found in abundance in seminal plasma. They regulate aspects of sperm cell function and are also thought to prevent immune-mediated destruction of sperm cells within the female reproductive tract. In a previous study, we isolated two distinct populations of prostasomes, differing both in size and protein composition, from the seminal fluid of vasectomized men. In the current study, we characterized the lipid content of these two prostasome populations. Both prostasome types had an unusual lipid composition, with high levels of sphingomyelin (SM), cholesterol, and glycosphingolipids at the expense of, in particular, phosphatidylcholine. The different classes of glycerophospholipids consisted mainly of mono-unsaturated species. The sphingosine-based lipids, SM and the hexosylceramides, were characterized by a near absence of unsaturated species. The two types of prostasome differed in lipid composition, particularly with regard to the relative contributions of SM and hexosylceramides. Potential implications of the lipid compositions of prostasomes for the mechanisms of their formation and function are discussed.

Comparative proteome analysis to explore p53 pathway disruption in head and neck carcinogenesis.

The 5-year-survival rate of head and neck squamous cell carcinoma (HNSCC) has been only moderately improved over the last few decades. HNSCC develops in precursor fields of genetically altered mucosal cells, typically characterized by p53 pathway disruption, that mostly do not give any clinical symptoms. Patients present therefore often with invasive carcinomas in an advanced stage. After tumor resection, part of these fields frequently stays behind unnoticed, causing secondary tumors. Identification of these precursor fields would allow screening and early detection of both primary and secondary tumors. Our aim was to identify differential proteins related to p53 dysfunction. These proteins may serve as valuable biomarkers that can predict the presence of a precursor field. We used a squamous cell model for p53 inactivation, which was analyzed by 2D-DIGE and LC-MS/MS. This approach enabled us to identify a set of 74 proteins that were differentially expressed in cells with normal versus disrupted p53 function. For six proteins the major changes in expression were verified with immunohistochemical staining. The most promising result was the identification of peroxiredoxin-1 which allowed immunohistochemical discrimination between normal epithelium and precursor field tissue with a TP53 mutation.