Abstract
Bioorthogonal Noncanonical Amino acid Tagging (BONCAT) is a technique first reported in 2006. This technique has rapid developed and widely used in the field of proteomics in the past decade. This technique employs the click chemistry (the clever combination of two substrates with specific biomolecules) and azidohomoalanine (AHA) (an azide-bearing noncanonical amino acid which can replace methionine in -translation and be tagged by a biotin-alkyne through “copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)” via click chemistry afterwards). These “biotinylated proteins” can further be analyzed by mass spectrometry (MS) or visualized by fluorescence microscopy. Many earlier studies have indicated that the above-described AHA-BONCAT does not harm cells when cells are incubated with AHA for proper periods of time and at mM concentrations. However, whether replacing the methionine in the medium by AHA may influent cell growth is still clear. In this study, I find that AHA incubations for periods loner than 4 hours cause cell death, slower growth rate and abnormal cell cycle. These results indicated that AHA incubation for periods longer than 2 hrs will produce negative effects on cells. Moreover, “Direct Detection of Biotin-containing Tags (DiDBiT)” is a modified version of BONCAT and has been developed to improve the identification of newly nascent AHA-containing peptides by MS analysis. Here, I find that this technique has limitations to be used to detect small amounts of sample, such as in search for nascent proteins in cultured rat cortical neurons. However, this method can help visualize newly synthesized proteins in cultured neurons.