SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins

Abstract

Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.

IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

“…The half-life of IL-2Rα was measured using a protocol designed by Morey, et al. ( 13 ) in which a traditional pulse-chase experiment was performed using L-azidohomoalanine (AHA), a methionine analog, as the pulsed label. Incorporation of AHA into nascent proteins was detected by a click chemistry based, copper-free strain-promoted alkyne-azide cycloaddition reaction in which a fluorescent cyclooctyne probe (dibenzocyclooctyne-488) bound to incorporated AHA ( 14 , 15 ).…”

“…The latter antibody was chosen for its strong signal and suggests that IL-2Rα is active. A linear regression analysis was then performed on normalized levels of labeled IL-2Rα and half-life was calculated via the following equation: t 1/2 = ln(2)/slope of decay ( 13 ).…”

IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells

“…The half-life of IL-2Ra was measured using a protocol designed by Morey, et al (13) in which a traditional pulse-chase experiment was performed using L-azidohomoalanine (AHA), a methionine analog, as the pulsed label. Incorporation of AHA into nascent proteins was detected by a click chemistry based, copper-free strain-promoted alkyne-azide cycloaddition reaction in which a fluorescent cyclooctyne probe (dibenzocyclooctyne-488) bound to incorporated AHA (14,15).…”

“…The latter antibody was chosen for its strong signal and suggests that IL-2Ra is active. A linear regression analysis was then performed on normalized levels of labeled IL-2Ra and half-life was calculated via the following equation: t1/2= ln(2)/slope of decay (13).…”

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

“…We used 4-azido-L-phenylalanine (4AZP) as the non-canonical amino acid and covalently attached DBCO-Cy3 using strain-promoted cyclo-addition click chemistry (Kumaresan et al 2000;Kuppa et al 2021;Bednar et al 2021;Jackson, Hammill, and Mehl 2007;Morey et al 2021). Detailed methods for generating Cy3-labeled hXPA (hXPA Cy3 ) are detailed below.…”