In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. The selectivity of the method can be increased by the use of mutant aminoacyl-tRNA synthetases (aaRSs) that allow the incorporation of ncAAs not used by the endogenous biomachinery.
The selectivity of the method can be increased through the use of mutant aminoacyl tRNAs. The lack of signal in cells in which protein synthesis is inhibited confirms the selectivity of the method regarding the labeling of newly synthesized proteins. Image of mammalian cells pulsed with Aha in the absence (top) or presence (bottom) of the protein synthesis inhibitor anisomycin.
Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows the incorporation of new amino acids into proteins in vivo. To validate this approach, we first confirmed that the incorporation of 2 into newly synthesized proteins is dependent on the expression of the mutant synthetase. Analysis of the lysate (L), unbound flow-through (FT), washes (W1, W3, W5) and eluent (E) reveal a separation of representative proteins from bacteria and mammals.
For example, transcription from the soxS promoter in Escherichia coli drives expression of the SoxS protein, a transcription factor responsible for.
NLL-MetRS expression, as revealed by western blot analysis (Fig. 3.2c), was consistent with the behavior of the SoxRS regulon and fluorescence analysis of protein labeling. When transcriptional activity of the poi is "off", the NLL-MetRS is not expressed and proteins are not labeled. Coomassie staining was used to confirm equal loading of protein between lanes (right). e) Western analysis of NLL-MetRS expression in uninduced (-) and induced (+) cells.
Proteomic labeling by Anl under conditions of oxidative stress. a) NLL-MetRS is under the control of the SoxRS regulon and is activated by superoxide or nitric oxide. NLL-MetRS is under the control of the soxS promoter and can be activated by superoxide or nitric oxide. The level of labeling in the SoxRS system depends on the level of induction of NLL-MetRS expression.
NLL-MetRS is under the control of the soxS promoter and is activated by the addition of paraquat (PQ) to the culture medium. The gene encoding NLL-MetRS was amplified from pQE80-NLL by PCR using primers: 5'- atatggatccatgactcaagtcgcgaaga-3' and 5'-ggtggaagcctctaaatgaagatct-3'. The cassette encoding NLL-MetRS under the control of the SoxRS regulon was amplified from pSOX_IDT_NLL by PCR using the primers: 5'-atatgctagccccgtgtaaaacgacggccagt-3' and 5'- atatgctagcagtattgagcctcaggaaac-3'.
The resulting fragment was inserted into the NheI site of the low copy number pACYC177 plasmid to form pSOX-NLL-MetRS. To one culture, arabinose was added to a final concentration of 0.2% for induction of NLL-MetRS. Membranes were probed for NLL-MetRS by adding Anti-PentaHis AlexaFluor647 conjugate to the blocking buffer at a dilution of 1:5,000.
Next, we asked whether NLL-MetRS was functional when expressed in the cytoplasm of mammalian cells. Expression of the NLL-MetRS-mCherry fusion was confirmed by fluorescence microscopy, western analysis and affinity. We assessed the activity of NLL-MetRS in HEK293-8D3 by searching for evidence of Anl incorporation into cellular proteins.
To assess the selectivity of the enrichment procedure, a "mock enrichment" of proteins from unlabeled cells was processed in parallel. Each step of the enrichment process was followed by coomassie staining of protein gels and streptavidin detection of biotinylated proteins (Fig. 4.2A-B). We did not detect coomassie-stained proteins in the elution of the mock enrichment (Fig. 4.2A, lane "E"), suggesting that proteins enriched from HEK293-8D3 cells (Fig. 4.2B, lane "E" ) contained very few (if any) labeled proteins.
While GAPDH and β-actin were absent in the mock enrichment elution, both were present in the enriched HEK293-8D3 protein fraction (Supplementary Figure 4.7). The N-terminal tryptic fragment of polypyrimidine tract binding protein (PTBP) was found to contain DIBAC-biotin-modified Na-acetylated Anl (Fig. 4.2C, Table 4.1). Thus, we were pleased to find that of the 14 peptides corresponding to PTBP internal fragments, none showed evidence of incorporation (four contained one or more Met residues).
In our early imaging experiments, we observed that a fraction of cells displayed increased fluorescence localized to the center of the cell. We verified that these proteins were histones by acid extraction of histones from nuclei and DIBAC-TAMRA labeling of the isolated proteins (Fig. 4.3B). A complete band shift of the azide-containing DHFR indicates that the reaction is highly efficient.
Enrichment was analyzed by Western blotting of the constitutively expressed “maintenance” proteins GAPDH and β-actin. Total sequence coverage of the associated protein is shown with individual peptides highlighted in yellow. TLC, 1H NMR and mass spectrometry were used to confirm the structure and purity of the amino acid.
The pulse was stopped by aspiration of the culture medium at which point cells were rinsed once with room temperature PBS. Reactions with DIBAC-TAMRA were performed at a final concentration of 20 µM of the probe and incubated for 20 m protected from light and with rotation at room temperature. Lysates were prepared as described above so that the final volume of the solution was ∼2 ml after alkylation.
We confirmed that the protein purified from HEK293-8D3 cells was indeed NLL-MetRS-mCherry by in-gel tryptic digestion of the band-containing gel fragment and LC/MS/MS analysis of the extracted peptides as described above. The expression of specific sets of proteins plays an essential role in defining both the identity and the functional state of the cell (Tomancak, 2007).
CONCLUDING REMARKS
