posted on 2023-12-19, 13:06authored byNicole Potter, Simon Latour, Edmond C. N. Wong, Mitchell A. Winnik, Hartland W. Jackson, Alison P. McGuigan, Mark Nitz
Mass
cytometry permits the high dimensional analysis of complex
biological samples; however, some techniques are not yet integrated
into the mass cytometry workflow due to reagent availability. The
use of self-labeling protein systems, such as HaloTag, are one such
application. Here, we describe the design and implementation of the
first mass cytometry ligands for use with HaloTag. “Click”-amenable
HaloTag warheads were first conjugated onto poly(l-lysine)
or poly(acrylic acid) polymers that were then functionalized with
diethylenetriaminepentaacetic acid (DTPA) lutetium metal chelates.
Kinetic analysis of the HaloTag labeling rates demonstrated that the
structure appended to the 1-chlorohexyl warhead was key to success.
A construct with a diethylene glycol spacer appended to a benzamide
gave similar rates (kobs ∼ 102 M–1 s–1), regardless
of the nature of the polymer. Comparison of the polymer with a small
molecule chelate having rapid HaloTag labeling kinetics (kobs ∼ 104 M–1 s–1) suggests the polymers significantly reduced the
HaloTag labeling rate. HEK293T cells expressing surface-exposed GFP-HaloTag
fusions were labeled with the polymeric constructs and 175Lu content measured by cytometry by time-of-flight (CyTOF). Robust
labeling was observed; however, significant nonspecific binding of
the constructs to cells was also present. Heavily pegylated polymers
demonstrated that nonspecific binding could be reduced to allow cells
bearing the HaloTag protein to be distinguished from nonexpressing
cells.