Quantification of amino acids secreted by yeast cells by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Monitoring the levels of amino acids (AAs) in biological cell cultures provides key information to understand the regulation of cell growth and metabolism. Saccharomyces cerevisiae can naturally excrete AAs, making accurate detection and determination of amino acid levels within the cultivation medium pivotal for gaining insights into this still poorly known process. Given that most AAs lack ultraviolet (UV) chromophores or fluorophores necessary for UV and fluorescence detection, derivatization is commonly utilized to enhance amino acid detectability via UV absorption. Unfortunately, this can lead to drawbacks such as derivative instability, labor intensiveness, and poor reproducibility. Hence, this study aimed to develop an accurate and stable hydrophilic interaction liquid chromatography-tandem mass spectrometry analytical method for the separation of all 20 AAs within a short 17-min run time. The method provides satisfactory linearity and sensitivity for all analytes. The method has been validated for intra- and inter-day precision, accuracy, recovery, matrix effect, and stability. It has been successfully applied to quantify 20 AAs in samples of yeast cultivation medium. This endeavor seeks to enhance our comprehension of amino acid profiles in the context of cell growth and metabolism within yeast cultivation media.

Isolation of Mitochondria from Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is an important model organism to study cellular structure and function. Due to its excellent accessibility to genetics and biochemical and microscopic analyses, studies with yeast have provided fundamental insights into mitochondrial biology. Yeast offers additional advantages because it can grow under fermenting conditions when oxidative phosphorylation is not obligatory and because the majority of mitochondrial structure and function are largely conserved during evolution. Isolation of mitochondria is an important technique for mitochondrial studies. This chapter focuses on procedures for the isolation and purification of intact yeast mitochondria that can be used for numerous functional assays as well as for analyses of mitochondrial ultrastructure.