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  • Chemical cytometry utilizes sensitive analytical techniques

    2020-08-04

    Chemical cytometry utilizes sensitive analytical techniques such as microelectrophoresis or mass spectrometry to analyze and characterize the contents of single GSK-2118436 (Dovichi, 2010; Dovichi & Hu, 2003). Microelectrophoretic chemical cytometry is well-suited to address many of the challenges of single-cell analysis by virtue of its low sample volume requirements (pL to nL), superb resolving power (hundreds of analytes), and extremely low detection limits (10mol), enabling separation of a large number of analytes from single cells with sub-pM detection limits (Vickerman, Anttila, Petersen, Allbritton, & Lawrence, 2018). These attributes, in combination with the absence of the need for cell genetic engineering, make this technique ideal for analyzing single cells from small mixed populations such as that from a primary clinical sample. Furthermore, chemical cytometry can provide a direct readout of enzyme activity, irrespective of the DNA, RNA, or protein levels in the cells, yielding valuable information about active cellular processes that cannot be obtained from genetic or expression information alone. However, a key challenge of this technique is design of a suitable probe that meets the strict requirements for reporting enzyme activity in single cells. These requirements include a probe or substrate with sufficient specificity to reliably report the activity of an enzyme or group of enzymes, the ability to load the probe into single cells without perturbing signaling pathways, and an intracellular lifetime sufficiently long to measure the desired process. Herein, we focus on the design of a reporter for chemical cytometry, specifically on the selection, optimization, and validation of intracellular reporters (typically short peptides) for protein kinase activity measurements by chemical cytometry.
    Target enzyme attributes and substrate peptide selection Protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues in both proteins and peptides using ATP as the phosphoryl donor. The human kinome is comprised of 518 protein kinases and 40 lipid kinases. The vast majority (478) of the former contain the so-called eukaryotic protein kinase (EPK) domain, a stretch of approximately 250 residues that encompass the catalytic region (Duong-Ly & Peterson, 2013; Kostich et al., 2002). The substrate specificity of the large EPK family is controlled by three key determinants: (1) The active site substrate specificity defines the ability of a protein kinase to phosphorylate one or more alcohol-bearing residues in the active site. Indeed, the EPKs are divided into three distinct groups based on their active site specificity: the tyrosine proteins kinases (TPKs), the serine/threonine protein kinases (SPKs), and the so-called dual specificity protein kinases that catalyze the phosphorylation all three types of alcohol-bearing residues (Miller & Turk, 2018). However, the active site specificity of protein kinases is not limited to the three genetically encoded alcohol-containing amino acids. A wide variety of unnatural residues are phosphorylated by protein kinases, including a structurally constrained tyrosine residue [(7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Htc)] (Kwon, Mendelow, & Lawrence, 1994; Kwon et al., 1993; Lee, Niu, & Lawrence, 1995; Prorok, Sukumaran, & Lawrence, 1989; Turner et al., 2016). Substrates containing Htc are particularly useful as probes of TPK activity since the corresponding phosphorylated product is resistant to dephosphorylation by intracellular protein phosphatases (Turner et al., 2016). One of the key advantages of using peptides as protein kinase substrates is that unnatural residues are readily introduced during peptide synthesis. Non-naturally occurring residues have been used to endow peptide-based substrates with useful properties, including enhanced selectivity for specific protein kinases, resistance to intracellular proteolysis (vide infra), and photo-transformation from inactive to active substrates.