Polyphosphate (polyP), a biopolymer composed of phosphates, impacts a wide range of biological functions and pathological conditions in all organisms. However, polyP’s intricate physiology and structure in human cells have remained elusive, largely due to the lack of a reliable quantification method including its extraction. In this study, we assess critical points in the whole process: extraction, purification, and quantification polyP from human cell lines. We developed a highly efficient method that extracts between 3 and 100 times more polyP than previously achieved. Supported by Nuclear Magnetic Resonance (NMR), our approach confirms that mammalian polyP is primarily a linear unbranched polymer. We applied the optimized method to commonly used human cell lines, uncovering important variations of intracellular polyP that correlate with the expression levels of specific polyP converting enzymes. This study underscores the importance of employing several techniques for polyP characterization in parallel and provides a valuable and standardized tool for further exploration in this field.
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