Design Parameters for a Mass Cytometry Detectable HaloTag Ligand.
Bioconjug Chem
; 35(1): 80-91, 2024 01 17.
Article
em En
| MEDLINE
| ID: mdl-38112314
ABSTRACT
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 â¼ 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 â¼ 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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Polímeros
/
Proteínas
/
Hidrolases
Limite:
Humans
Idioma:
En
Revista:
Bioconjug Chem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Canadá