Alkyl-Chain; Methionine analogue.

The E3 ubiquitin ligase ligand and the target protein ligand are the two distinct ligands found in PROTAC, which are joined by a linker. Specifically, target proteins are degraded by PROTAC through the intracellular ubiquitin-proteasome system [2].

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In order to facilitate the click-chemistry enrichment of a small sample of proteins being synthesized in Arabidopsis plants or cell cultures for analysis, homopropargylglycine enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT)[1].

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Effect of Met, AHA, HPG and 15N labelling on plant cell growth [1]
To begin a systematic comparison of BONCAT labelling in plant cell culture, we examined the effects of AHA, HPG and 15N on cell culture growth. Met and the ncAAs were effectively taken up into Arabidopsis cells, as determined by targeted LC-MS (Table 1). The recorded cell content of ncAAs correlated with a loss of cell growth rate in AHA-treated cells, but no effect on the growth rate of 15N-treated cells was observed; a less severe decrease in growth rate was also observed in HPG-treated cells (Figure 1d). Assessment of cell death showed HPG caused less cell death than AHA in Arabidopsis cell cultures (Figure 1b).

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AHA- and HPG-stimulated in-vivo synthesis of Met and related metabolites [1]
To reveal the possible cause of the cell culture growth inhibition caused by AHA or HPG, we determined changes in the levels of Met and four related metabolites in Arabidopsis cell cultures treated with AHA, HPG or Met over the course of 24 h (Figure 2). After 24 h there was no significant difference between the level of S-adenosylhomocysteine, homocysteine or SAM (P > 0.05), but the levels of 5-methylthioadenosine were significantly higher (P < 0.05) in the cells treated with Met and AHA and, to a lesser extent, in cells treated with HPG. The differences in the average level of Met after 24 h appeared different, but the large standard deviation rendered this difference not statistically significant. Nevertheless, the observed disruption of Met metabolism may be the cause of effects on cell growth that we observed in cell cultures treated with AHA and the less dramatic effects observed in cell cultures treated with HPG. After 24 h of treatment, the ratio of the supplied ncAA to endogenous Met was 1.6 for AHA and 4.8 for HPG (Table 1 and Figure 2).

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HPG was more efficiently incorporated into proteins replacing Met than AHA [1]
After establishing that HPG caused less metabolic disruption that AHA in Arabidopsis cell cultures, we used LC-MS/MS to determine the number of proteins tagged with AHA or HPG after 24 h of exposure. We identified a similar total number of protein groups in AHA- and HPG-treated cells (3177 and 3146 protein groups, respectively) using data-dependent acquisition proteome profiling (Tables S4 and S5). However, in the case of treatment with AHA, none of the proteins showed any peptide MS evidence of AHA replacing Met in the amino acid sequence of identified peptides. In contrast, 100 of the protein groups identified in the cells treated with HPG (3.2%) showed MS evidence of HPG replacing Met in peptides matching their amino acid sequences (Table 2, Figure 3). Thus, it was clear that HPG was a much more effective ncAA for tagging a sample of nascent proteins, not only because it caused less metabolic disruption and less cell death, but also because tagging of proteins could be verified even in whole proteome extracts analysed by data-dependent acquisition. Proteomics datasets from AHA- and HPG-treated cells were inseparable by principal components analysis (PCA), but both of these datasets were slightly separated from proteomics datasets from untreated cells, indicating that AHA and HPG treatments alone have some effect on the plant cell proteome composition (Figure 4a).

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Enrichment of AHA- and HPG-tagged proteins from plant cells [1]
When AHA-tagged proteins from plants were previously reported (Glenn et al., 2017; Yu et al., 2020), proteins were cleaved off the enrichment resin using trypsin, which could result in the ncAA tags remaining on the resin and thus not appearing in peptide MS data. However, this lack of direct evidence of AHA-tagging of proteins also leaves open the possibility that proteins from AHA-treated plants may become enriched through alternative plant-specific and/or yet-to-be-defined chemical interactions with the enrichment media, rather than through azide–alkyne cycloaddition.

To verify the specificity of the enrichment of click-chemistry products in our experiments, we enriched intact proteins from cell cultures treated with HPG and AHA (Table 2, Tables S4 and S5). AHA enrichment uses alkyne functionalized resin while HPG enrichment uses azide functionalized resin. PCA showed that the HPG enrichment processes produced protein sets of composition and intensity-based absolute quantification (iBAQ) abundances that were distinct from the total protein samples extracted from cell cultures (Figure 4b). In contrast, the enriched and bulk datasets from AHA-treated cell cultures were inseparable by PCA (Figure 4c). Interestingly, PCA also showed that the HPG- and AHA-enriched datasets were distinct from each other (Figure 4d). Previously, it has been shown that enrichment of untreated proteins using this chromatography approach is negligible as it is performed under very stringent conditions with high urea concentrations used for washing and large dilution volumes that will prevent any significant non-covalent associations from being retained (Zhang et al., 2014).

To determine if the enrichment was selective for proteins with the HPG and AHA tag, we used MS to look for the presence of Met-to-HPG and Met-to-AHA substitutions in peptides from bulk proteins from the ncAA-treated cells and compared them with peptides from proteins that were detected in the protein samples after enrichment. The results of these experiments are summarized in Table 2.

For the cell cultures treated with HPG, 555 protein groups were found in the resin-enriched protein samples, and 461 of these were also found in whole tissue extracts; thus approximately 15% of the 3146 protein groups identified in whole tissue extracts were also identified in the enriched dataset. Of the 555 enriched protein groups, 74 showed evidence of HPG tagging in the bulk dataset (Table 1, Table S6). We also calculated a relative enrichment value ([riBAQ enriched – riBAQ total]/[riBAQ enriched + riBAQ total]); a value >0 indicates a protein with a higher relative abundance in the enriched than the total extract. Within the 555 enriched protein groups, 240 (43%) had an enrichment value >0 and of these, 39 showed evidence of HPG tagging in the bulk dataset (Table 1, Table S6). It should be noted that not all proteins would produce a peptide with an ncAA that is also detectable by LC-MS/MS, so one would expect that proteins for which no tag can be experimentally detected could increase in abundance during the enrichment process. Proteins with enrichment values <0 may be due to a low stoichiometry of HPG tags or weak binding of a non-tagged protein. Only seven of the 555 protein groups showed experimental evidence of HPG tagging in the resin-enriched protein samples, but this could be explained by cleavage of proteins from the enrichment resin with trypsin, leaving many HPG-tagged peptides still attached to the resin.

In comparison, for cell cultures treated with AHA, only 131 protein groups were found in the resin-enriched protein samples. Of these, 78 protein groups (60%) had an enrichment value >0, even though there was no evidence of AHA tagging of peptides for any of these proteins in the bulk or resin-enriched datasets. Comparing the absolute numbers of protein groups in the enriched datasets from both treatments might suggest that AHA is more selectively incorporated into nascent proteins than HPG. However, comparing the percentage of the enriched proteins with enrichment values >0 between the two treatments suggests that AHA may be incorporated to a greater extent than HPG in the proteins that it tags. Notwithstanding the inefficiency of the method, the trends described above were also seen in experiments where the proteins were cleaved from the resin using hydrazine and where peptides that remained bound after on-resin digestion were cleaved from the resin using hydrazine and then separately analysed (Table 2).

Taken together, these results suggest that the click-chemistry enrichment process is somewhat opaque and proteins that are bound through interactions other than azide–alkyne cycloaddition with ncAAs may be contributing to the protein sets identified. Therefore, we caution against the notion that the presence of a given protein in a click-chemistry–enriched protein sample is sufficient to conclude that this protein has been tagged with ncAAs. Rather, enrichment should be confirmed by MS evidence of ncAA incorporation in peptides before or after enrichment to verify members of a nascent protein set. Furthermore, the data from our HPG-treatment experiment show that not all tagged proteins bind effectively to the resin (our data show that 26 of the 100 Met-to-HPG–tagged proteins from the HPG-treated cells were not identified in the alkyne-resin enriched dataset), so there appears to be some unidentified bias in the enrichment process that causes the loss of some bona fide tagged proteins.

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AHA and HPG tag some of the same proteins as 15N [1]
To verify how well the ncAA tagged sample protein populations represented the entire nascent protein pool, we compared them with proteins labelled by the incorporation of 15N salts into proteins over 24 h. Of the 100 protein groups that were found tagged with HPG, 62 were also found in the 366 protein groups that were significantly labelled with 15N in a 24 h period in cell cultures (Figure 3). To order the 15N-labelled proteins according to their abundance of nascent protein, we multiplied the 15N-labelled protein fraction by the abundance of each protein from untreated cell culture datasets. This abundance was estimated using iBAQ. When we sorted the protein groups list from most to least newly synthesized protein we found that 34 of the top 40 protein groups in this list were also verified as HPG tagged (15) and/or resin-enriched from HPG-treated samples (32) (Figure 5, Table S6). While 27 of the top 40 protein groups were also resin-enriched from AHA-treated samples, none was verified as AHA tagged (Figure 5, Table S6).

Comparison with labelling by 15N provided several avenues to investigate the selectivity of the ncAA enrichment process for nascent proteins. There was a substantial overlap between the protein groups that were labelled with 15N and those that were detected in the resin-enriched samples from cells treated with AHA or HPG. Of the 366 protein groups that were tagged with 15N, 90 and 219 of these were also enriched in the samples from the cells treated with AHA or HPG, respectively (Figure 4e; 25% and 60% of the protein groups labelled with 15N were also present after enrichment in samples from cell cultures treated with AHA or HPG, respectively). However, only 69 protein groups were shared between all three lists (Figure 4e); 21 enriched groups were shared only between the 15N and AHA enriched dataset and 150 enriched groups were shared only between the 15N and HPG enriched dataset. These overlaps decreased substantially when only protein groups with enrichment values >0 were considered (Figure 4f).

Enrichment is weakly linked to percentage MET, but not to protein length, number of tags per protein, relative position of tags or abundance of newly synthesized protein As AHA or HPG tags proteins in the position of Met residues, it is important to clarify if enrichment was significantly biased by the protein percentage Met, length, number of tags or relative position of tags within amino acid sequences. We looked for correlations between each of these attributes and the enrichment values (see Figures S1–S6). The only correlation that was apparent was a weak one between the percentage of Met in a protein and its relative-enrichment (Figures S1 and S2); this correlation was stronger in the case of cells treated with HPG, compared with those treated with AHA. It might be expected that the proteins displaying the highest enrichment would be those with the highest abundance of newly synthesized material based on 15N labelling; however, we found no such correlation (Figures S7 and S8) for enrichment using either ncAA tag.

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Gene Ontology term enrichment analysis reveals the types of proteins that are tagged and enriched by 15N and ncAAs [1]
Using AgriGO (Du et al., 2010; Tian et al., 2017), we observed some degree of Gene Ontology (GO) term enrichment in tagging and ncAA enrichment sets. HPG and 15N both favoured tagging of stimulus response proteins, but for HPG they were mainly heat response proteins while for 15N they were mainly cadmium response proteins (Figures S9 and S10). In terms of click-chemistry enrichment, proteins resin-enriched from AHA-treated cells were those involved in carbohydrate metabolism and stress/stimulus response proteins (Figure S11), while stress and heat response proteins were overrepresented in samples resin-enriched from HPG-treated cells (Figure S12).
Taken together, these data indicate that stress/stimulus response proteins undergo rapid synthesis in Arabidopsis cell cultures. This is probably not a result of the treatment with ncAAs, as stress/stimulus response proteins were also overrepresented in the 15N-labelled dataset and there is no precedent for thinking 15N would cause synthesis of these types of proteins to be upregulated.

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Open mass searches did not reveal modifications of peptides from AHA-treated cells [1]
A possible reason for the lack of observable AHA tags in our data is that incorporation of AHA into plant proteins causes an unexpected mass change [e.g. through post-translational modification (PTM) of the AHA moiety]. To investigate this possibility, we performed open-mass searches on the data from AHA-treated and untreated cells. We used the Arabidopsis Genome Initiative locus codes from the enriched datasets and the HPG-tagged dataset to filter the data and then used the filtered data to construct frequency histograms for the mass modifications of peptides to look for evidence of new delta mass shifts following AHA incorporation, independent of a tight precursor mass filter for AHA. However, there was no clear evidence from the comparison of delta mass shift histograms for novel mass shifts associated with AHA treatment of cells (Table S7).

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Development of a high confidence set of enriched, newly synthesized proteins [1]
From our combined data, we generated a high confidence list of 248 nascent proteins (Table 3; Table S8) that includes proteins that were enriched by click chemistry after treatment with AHA and/or HPG and (i) found to be tagged with HPG before enrichment, and/or (ii) are known to be independently labelled with 15N during the same time period. Using mapman functional categories as a guide (Schwacke et al., 2019), these proteins were classified into eight supergroups. About one-quarter of this sample population were involved with protein biosynthesis. Proteins involved in nucleotide metabolism and processing, cellular organization, nutrient uptake, photosynthesis, redox homeostasis, secondary metabolism, solute transport and vesicle trafficking and enzymes and coenzymes also featured in this list. The HPG enrichment population included more proteins than the AHA enrichment population for every supergroup of proteins examined.

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PTM profiles of nascent proteins differ to total protein sets [1]
Being able to resin-enrich a subset of nascent proteins enabled us to make some important observations about the differences between nascent protein extracts and total protein extracts. We analysed the data for 10 common PTMs (see Experimental procedures) and found that peptides of 55% and 51% of the proteins in total protein extracts from cell culture treated with AHA or HPG, respectively, contained one or more PTMs (Figure 6). In stark contrast, peptides from only 9% or 10% of proteins in resin-enriched nascent protein datasets from AHA- or HPG-treated cells, respectively, contained PTMs (Figure 6). Of the 214 proteins found in both nascent and total datasets using AHA, only three (1%) contained peptides with more PTMs in the enriched dataset than in the bulk dataset. Correspondingly, of the 593 proteins found in both nascent and total datasets using HPG, only nine (2%) contained more PTMs in the enriched dataset than the bulk dataset (Table S9). When individual proteins were considered, the proportion of spectral hits for PTM-containing peptides per protein was greater in the bulk datasets compared with their enriched counterparts in all cases for datasets from AHA-treated cells [25 protein IDs; P < 0.05 (n = 3); Table S9] and in all but three cases for datasets from HPG-treated cells [64 protein IDs, total; P < 0.05 (n = 3); Table S9]. Considering the larger HPG-treated dataset, the proteins that were significantly more modified in the bulk dataset compared with the nascent dataset were generally proteins related to primary metabolism or protein biosynthesis, homeostasis and modification. The high degree of modification of proteins in these pathways as they age may indicate that they are exposed for longer or to harsher (bio)chemical environments than other proteins in vivo.
We further analysed the data to see any differences in the relative frequency of occurrence of the 10 modifications across the nascent and bulk proteins from AHA- and HPG-treated cells (Figure 6, Table S9). This showed that the higher frequency of PTMs in the bulk protein extracts was largely due to higher frequency of Glu methylation, Met oxidation and Thr/Tyr phosphorylation.
Considering the AHA datasets, aside from small differences in oxidation (higher frequency in bulk dataset) and N-terminal acetylation (higher frequency in nascent dataset), the nascent and bulk proteins appeared similar in terms of PTM frequencies (Figure 6b). On first analysis it appears that there is a very high proportional frequency of Arg methylation in nascent proteins from AHA-treated cell cultures (Figure 6), but, of the 129 Arg-methylated peptide spectra found in this dataset, all but two were mapped to an heat shock protein (HSP)70 chaperone (AT3G09440.1), indicating that this is not a general difference between bulk and nascent protein sets but a case of enrichment of a specific protein with a dominant PTM.
In contrast, nascent proteins from HPG-treated cell cultures contained a much lower frequency of MET oxidation and a much higher frequency of N-terminal acetylation compared with the matched bulk protein sets (Figure 6c). The N-terminal acetylation was found across 48 HPG-enriched proteins. The set of nascent proteins with lower number of spectral counts to PTM containing peptides included major proteins involved in the ribosome, respiratory metabolism and molecular chaperones (Table S9).

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue Homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15 N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants [1].

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HPG/Homopropargylglycine is more effective than AHA for protein tagging in whole plants [1]
Whole Arabidopsis plants treated with AHA via seedling flood, have been previously reported to show incorporation of AHA into proteins using specific antibodies, and separately, proteins enriched using click chemistry have been identified using peptide MS (Glenn et al., 2017; Yu et al., 2020). While these studies show the general utility of BONCAT, neither provided MS/MS evidence of AHA residues replacing Met residues in tagged proteins identified by MS. To determine the extent of AHA labelling in AHA-treated plants, we treated Arabidopsis plants with AHA using the same technique. Proteins isolated from plants treated with AHA had MS-confirmed AHA tags in only one of the 3022 protein groups (0.03%; one tagged peptide was identified) in total extracts of whole seedlings (Figure 1a, Table S2). As an independent comparison we also treated plants with the alternative ncAA, HPG, using the same method and, in contrast, these plants displayed HPG tags in 11 of 2917 protein groups (0.37%; 16 unique tagged peptides were identified; Figure 1a, Table S3). Therefore, HPG appeared to be more effective than AHA for tagging a sample population of nascent proteins in whole plants, although the seedling flood in our hands appeared to have limited use for studying protein synthesis in whole plants. Assessment of green leaf area viewed from above showed that flooding with AHA caused inhibition of growth after 24 h but flooding with HPG did not; this demonstrates that HPG might be a more suitable BONCAT agent for whole plants. Nevertheless, after 48 h, AHA-treated and, to a lesser extent, HPG-treated plates both showed retardation of growth (Figure 1c). To investigate further the potential use of AHA and HPG as nascent-protein–tagging agents in plants, we therefore turned to a series of experiments using Arabidopsis cell culture where a flooded environment is the control state of cells.

Click-chemistry enrichment of AHA/HPG tagged proteins [1]
For experiments where enrichment of azide- (AHA) or alkyne- (Homopropargylglycine/HPG) tagged proteins was required, Click Chemistry Tools Click-&-Go Protein Enrichment Kits were used, with minimal modifications to the protocol. Tissue samples (1–2 g) were extracted as described above (except that the precipitation and washing step volumes were increased seven-fold) up to completion of the acetone wash steps. After this point, the pellets were resuspended in 3.2 ml of Lysis Buffer and the protein concentrations determined using Amido Black assays. The click reaction was performed as described in the protocol, except the reaction mixtures were prepared as follows: protein solution: 2400 µl (containing 1 mg of protein), 2× Cu catalyst solution: 3000 µl, resin: 200 µl, H2O: 400 µl. After the resin-bound proteins were washed, the resins were each split approximately in half, and one half of each was treated with trypsin and the other was treated with hydrazine hydrate (2% v/v solution) and then washed with phosphate-buffered saline (containing 1% w/v SDS, as recommended by the supplier, although the protocol does not call for SDS in this solution) to cleave the bound proteins from the resin. The resin that had been treated with trypsin was then also treated with hydrazine hydrate (2% v/v solution) to cleave the peptides that remained bound to the resin after digestion. The proteins that had been cleaved from the resin with hydrazine were precipitated using ice-cold acetone (≥10× solution volume). The resulting protein pellets were resuspended in digestion buffer (according to the protocol) and digested with trypsin. The enriched samples that were cleaved from the resin (both those that were cleaved off as whole proteins and those that were cleaved after trypsin digestion) were not fractionated, rather residual SDS and salt was removed by high-performance liquid chromatography using a J4-SDS column mounted to a C18 trap. These peptide samples were purified further, along with the peptides that had been generated by digestion of the resin-bound proteins, using silica C18 MacroSpin Columns. Clean peptide fractions were dried under reduced pressure and stored at −80 or −20°C until they could be analysed.

Plant growth conditions [1]
Sterilized (70% v/v ethanol in H2O with 0.05% v/v Triton X100 rotating end-over-end for 5 min before being rinsed with 100% ethanol) Arabidopsis thaliana (Col-0) seeds were sprinkled on to agar plates (0.9% w/v agar, half strength Murashige & Skoog salts, 0.3% sucrose). The seeds were stratified for approximately 2 days before being transferred into continuous light (approximately 600 µmol m−2 sec-1) at 22°C. Three-day-old seedlings were treated with AHA or Homopropargylglycine/HPG (1 mm in 9.9 mm potassium citrate buffer (pH 5.6, with 150 mm sucrose, 4.32 mg ml−1 Murashige & Skoog salts and 0.0024% v/v Silwett L-77) via seedling flood for 2 min (Glenn et al., 2017). The solutions were decanted and the plants harvested 3 h later into liquid N2.

To determine the longer-term effects of ncAA treatment on Arabidopsis growth, the treatment procedure outlined above was repeated, except the plants were allowed to continue growing for 72 h after treatment (in the same conditions). Two plates were subject to each treatment. Plants were photographed immediately after treatment and then again after 24, 48 and 72 h. Photographs were analysed using the ‘Threshold Colour’ plug-in in ImageJ as described by Corral et al. (2017). To control for differences in the total number of germinated seeds on each plate, the total number of green pixels in each photo were normalized as follows: the initial green areas for each replicate were averaged and a factor was generated by dividing this average by the initial green area for each replicate. Each measurement was then multiplied by this factor to generate the normalized green area.

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Measuring the effect of AHA, Homopropargylglycine/HPG and 15N on plant cell growth [1]
Three-day-old Arabidopsis cell cultures (PBS-D) in liquid MSMO media (120 ml each), grown in the dark with constant agitation at 26–28°C, were treated with 1 mm AHA or HPG. Initial treatments were performed by adding the solid powders directly to the cell cultures to give the desired concentration. Follow-up experiments were conducted by adding enough filter-sterilized AHA or HPG solution (100 mm in H2O) to give final concentrations of 1 mm. Samples (each approximately one-third of the total culture volume) of cell cultures were harvested by vacuum filtration after approximately 0, 8 and 24 h of exposure to AHA, HPG or 15N. The fresh weight of each sample was recorded before snap-freezing in liquid N2. The fresh weights of a control were also obtained at these time points. Three replicates, each consisting of a separate cell culture flask, were used in each treatment. Small (<1 ml) samples of one replicate for each treatment were also taken for viability staining using propidium iodide and fluorescein diacetate followed by microscopic analysis.

[1]. In vivo homopropargylglycine incorporation enables sampling, isolation and characterization of nascent proteins from Arabidopsis thaliana. Plant J. 2021 Aug;107(4):1260-1276.

[2]. Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs. EBioMedicine. 2018;36:553-562.

[3]. Protocol for visualizing newly synthesized proteins in primary mouse hepatocytes. STAR Protoc. 2021;2(3):100616. Published 2021 Jun 17.

(2S)-2-azaniumylhex-5-ynoate has been reported in Cortinarius claricolor with data available.

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Here, we have followed up on these reports to understand better how AHA affects plant growth, to clarify which proteins are tagged by AHA and to compare AHA with the alternative BONCAT reagent Homopropargylglycine/HPG and to 15N-labelling of endogenous amino acid pools to understand more fully the utility of BONCAT for sampling nascent proteins in plant science. We conclude that BONCAT is a useful tool for sampling of nascent proteins in plants but HPG has better utility than AHA for labelling and isolation of nascent plant proteins for a variety of applications in the general sampling of plant protein synthesis, as well as for identification and/or isolation of tagged proteins for independent study.[1]

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BONCAT is a valuable tool for sampling nascent proteins in plants for further study, but it suffers from technical challenges that have not been encountered in other organisms, potentially associated with Met synthesis by plants. Using HPG/Homopropargylglycine rather than AHA appears to overcome some of these challenges. However, to realize the potential of this technology fully, further technical advancements are required, so that BONCAT can be applied to intact plants with greater efficiency that has been found to date (Figure 1). Furthermore, as questions remain about the selectivity of the click-chemistry method for enriching plant proteins, independent evidence from ncAA residue analysis or isotope labelling is currently needed to confirm claims. Nevertheless, we are confident that the use of BONCAT will enable new features of nascent plant proteins to be discovered, such as the accumulation of PTMs, to investigate proteins as they age in vivo. [1]

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Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15 N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants. [1]

C6H10CLNO2

163.60

163.04

C, 44.05; H, 6.16; Cl, 21.67; N, 8.56; O, 19.56

942518-19-6

L-Homopropargylglycine;98891-36-2;Homopropargylglycine;215160-72-8

136212900

White to off-white solid powder

3

3

3

10

144

1

Cl[H].O([H])C([C@]([H])(C([H])([H])C([H])([H])C#C[H])N([H])[H])=O

XGBWOGGSQVMSRX-JEDNCBNOSA-N

InChI=1S/C6H9NO2.ClH/c1-2-3-4-5(7)6(8)9;/h1,5H,3-4,7H2,(H,8,9);1H/t5-;/m0./s1

(2S)-2-aminohex-5-ynoic acid;hydrochloride

942518-19-6; (2S)-2-aminohex-5-ynoic acid hydrochloride; 832-354-4; L-Homopropargylglycine hydrochloride; L-Homopropargyl Glycine (hydrochloride); (S)-2-AMINOHEX-5-YNOIC ACID HYDROCHLORIDE; L-Homopropargylglycine (hydrochloride); (2S)-2-aminohex-5-ynoic acid;hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~125 mg/mL (~764.06 mM)
H2O :~100 mg/mL (~611.25 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (12.71 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (12.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (12.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)