Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)

Abstract

In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We describe here a technology, based on the cotranslational introduction of azide groups into proteins and the chemoselective tagging of azide-labeled proteins with an alkyne affinity tag, to separate and identify, specifically, the newly synthesized proteins in mammalian cells. Incorporation of the azide-bearing amino acid azidohomoalanine is unbiased, not toxic, and does not increase protein degradation. As a first demonstration of the method, we report the selective purification and identification of 195 metabolically labeled proteins with multidimensional liquid chromatography in-line with tandem MS. Furthermore, in combination with leucine-based mass tagging, candidates were immediately validated as newly synthesized proteins. The identified proteins, synthesized in a 2-h window, possess a broad range of biochemical properties and span most functional gene ontology categories. This technology makes it possible to address the temporal and spatial characteristics of newly synthesized proteomes in any cell type.

Fig. 1.

AHA is not toxic to mammalian cells. (A) HEK293 cells were incubated for 2 h with HBS, 4 mM methionine in HBS, or 4 mM AHA in HBS and then incubated with propidium iodide (PI) to stain for dead cells. Shown are Nomarski (Upper) and PI (Lower) signals. (Scale bar = 100 μm.) (B) Dissociated hippocampal cultured neurons (12 days in vitro) infected with a destabilized and myristoylated variant of GFP were incubated for 2 h with equimolar concentrations of AHA or methionine. Images show representative neurons expressing the GFP reporter indicating no change in the gross morphology of AHA-treated neurons compared to methionine controls. Arrows indicate dendrites, and arrowheads point to somata. (Scale bar, 50 μm.)

Fig. 2.

Protein synthesis-dependent incorporation of AHA and analysis of global protein synthesis levels in the presence of AHA. (A) Western blot analysis for biotinylated AHA-labeled proteins in cell lysates from HEK293 cells incubated with AHA in the absence or presence of the protein synthesis inhibitors anisomycin or cycloheximide as compared to methionine control. Biotin immunoreactivity depends completely on protein synthesis and the presence of the AHA. (B) Line scan of radioactivity-labeled newly synthesized proteins transferred onto a nitrocellulose membrane from cells treated with AHA (black line) or methionine (red line) for 2 h.

Fig. 3.

Newly synthesized AHA-labeled proteins are not subject to increased protein degradation. (A) Autoradiogram analysis of immunoprecipitated ubiquitinated newly synthesized proteins of cell extracts from AHA-treated HEK293 cells as compared to methionine control samples (2-h treatment in the presence of [³⁵S]cysteine). Quantification of radioactive signals of immunoprecipitates is shown in the graph. n = 11 in four independent experimental sets. (B) Corresponding Western blot analysis for ubiquitinated proteins of immunoprecipitates in AHA-treated HEK293 cells as compared to methionine control samples.

Fig. 4.

Purification of AHA-labeled proteins after azide–alkyne ligation with a biotin-FLAG-alkyne tag. (A) Structure of the trypsin-cleavable biotin-FLAG-alkyne tag 1. Biotin (red rectangle), alkyne (green rectangle), and the tryptic cleavage sites (blue scissors) are indicated. The FLAG epitope DYKDDDDK is separated from the biotin moiety by a short linker (GGA). (B) Western blot analysis for affinity-purified biotinylated proteins using the biotin-FLAG-alkyne tag. Cell lysates from both AHA and methionine-treated HEK293 cells were subjected to [3 + 2] cycloaddition with the biotin-FLAG-alkyne tag and subsequently purified by using Neutravidin affinity matrix. Except for the nonspecific protein staining of samples containing Neutravidin affinity resin (Neutravidin affinity-matrix bound), control samples show no biotin signal. Note the higher migration level of alkyne-tagged HA-HAP1A protein (lanes indicated by asterisks) compared to the untagged protein in the methionine control and in the supernatant of the AHA sample. Sizes of marker proteins are indicated. NA, Neutravidin.