QUANTITATIVE ANALYSIS OF LACTOYLGLUTATHIONE: A NEW 2D-HPLC-MS/MS APPROACH WITHOUT ION-PAIR AGENTS

Lysine lactylation (KLa) is a recently identified post-translational modification (PTM) with significant implications in cancer. KLa occurs on both histone and non-histone proteins and may involve enzymatic or non-enzymatic mechanisms, utilizing Lactyl-CoA and Lacotylglutathione (LGSH) as donor of the lactyl moiety, respectively [1-2]. The specific regulatory enzymes that mediate enzymatic lactylation remain still unclear, although acetyl-synthetase (ACSS2), acetyl-transferases (p300), and deacetylases (HDAC1-3) have also been proposed as potential mediators for histone lactylation [3-5]. The glyoxalase 1 (GLO1) pathway involved in LGSH generation is instead engaged in the non-enzymatic lactylation strategy. Interestingly, the lactylation of histone proteins provides additional evidence, linking tumor metabolic reprogramming to epigenetic regulation and transcriptional changes. In prostate cancer (PCa), lactate fuels mitochondrial metabolism and promotes epigenetic changes, primarily through histone acetylation [6]. However, the role of histone lactylation in PCa remains largely unexplored. Published method for the determination of LGSH use an high performance liquid chromatography coupled to tandem mass spectrometry (HLPC-MS/MS) system with the support of an ion coupling agent [7] which have a positive effect on the retention and chromatographic separation of polar and watersoluble analytes such as LGSH, but strongly negatively affect the detection sensitivity in mass spectrometry. The aim of this work was to develop a high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method for the determination of LGSH in PCa cells extracts, without the support of the ion-pair agents. In order to analyze the minimal variations in concentration of LGSH, we used a two-dimensional (2D) HPLC system coupled to a LTQ-Orbitrap, equipped with an ESI source. The development of the method involved both the optimization of the chromatographic system and the MS/MS acquisition. The use of 2D chromatography allowed to eliminate most of the polar molecules, during the loading step of injected sample, and to minimize the ion suppression effect on the analyte detection. MS/MS studies were conducted to select the best fragmentation conditions (collision energy and activation time). Furthermore, the selected product ions were analyzed and characterized in high resolution mass spectrometry (HRMS at 30k FWHM) in order to guarantee the selectivity of the method. LGSH was quantified in sample extracts of lactate-treated and untreated PCa cells. The obtained results show that lactate exposure in PCa cells significantly increased LGSH levels.