Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these N⁶-substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, Dictyostelium discoideum. Within Dictyostelium, CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the Dictyostelium life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that Dictyostelium produces 6 CK forms (cis-Zeatin (cZ), discadenine (DA), N⁶-isopentenyladenine (iP), N⁶-isopentenyladenine-9-riboside (iPR), N⁶-isopentenyladenine-9-riboside-5′ phosphate (iPRP), and 2-methylthio-N⁶-isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during Dictyostelium development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in Dictyostelium, this research offers new insight into the nature of CK biosynthesis, secretion, and function during Dictyostelium growth, development, and spore germination.

Electrospray ionization mass spectrometry (ESI‐MS) is a powerful tool for identifying and characterizing organometallic and coordination compounds. However, detection of fragile structures bound by weaker intermolecular forces can be significantly limited in ESI‐MS owing to the use of relatively harsh instrument conditions and configurations. In this study, a set of tests was developed to assess the softness of ESI‐MS systems. Two variants are presented: positive ion mode, utilizing a mixture of sodium ions and triphenylphosphine oxide producing [Na(OPPh₃)_n]⁺ ions (n=1–4), and negative ion mode utilizing Pd(PPh₃)₄ and sulfonated triphenylphosphine producing [Pd(L)(PPh₃)_n]⁻ ions (n=0–2), where softer instrument conditions preserve a higher proportion of the high‐coordinate ions and harsher conditions will result in increased detection of products of ion fragmentation. The results revealed notable variations in instrument softness, which were influenced by a combination of instrument design and experimental parameters. Meticulously optimizing experimental conditions and ESI‐MS parameters is essential to achieving the softest ionization possible, ensuring reliable analysis where applicable. This study offers valuable insight through straightforward tests that can be employed to assess the suitability of an instrument for specific research needs.