In Figure 1 the results of the different conditions are shown. Figure 1A (odd lanes for BSA, even lanes for fetuin) shows the results for different β-elimination procedures, in which the linkage between sugar and amino acid side chain is cleaved by an elimination-type reaction. The conditions employed are the commercially available β-elimination kit (lanes 2 and 3), NaIO4 oxidation followed by β-elimination (lanes 4 and 5),21 and β-elimination with HNMe
2 (lanes 6 and 7).20 Figure 1B shows the results obtained with TFMSA at either 4 ºC or room temperature (lanes 10 and 11, respectively, for fetuin; lanes 12 and 13 for BSA).24 Anisole was added as a scavenger to keep the side chains of the amino acids intact.26
Figure 1. SDS-PAGE analysis of different deglycosylation conditions. A: (1) Fetuin; (2) Fetuin, β- elimination kit; (3) BSA, β-elimination kit; (4) Fetuin, NaIO4 o.n., 4 h pH 10.5; (5) BSA, NaIO4 o.n.,
4 h pH 10.5; (6) Fetuin, HNMe2, 55 ºC, 6 h; (7) BSA, HNMe2, 55 ºC, 6 h; (8) BSA. B: (9) Fetuin;
(10) Fetuin, TFMSA/anisole (3/2), 1 h, 4 ºC; (11) Fetuin, TFMSA/anisole (3/2), 1 h, r.t.; (12) BSA, (13) BSA, TFMSA/anisole (3/2), 1 h, 4 ºC; (14) BSA, TFMSA/anisole (3/2), 1 h, r.t.
In short, the β-elimination kit gave hardly any deglycosylation, and showed some degradation of both fetuin and BSA; β-elimination using HNMe2 showed complete protein loss for fetuin and heavy protein backbone degradation for BSA. On the other hand, NaIO4 oxidation, followed by β-elimination, showed no protein degradation, but also incomplete deglycosylation of fetuin, as indicated by the broad band in Figure 1A, lane 4. Likely, the last procedure only removed the O- linked glycans, while keeping the N-linked glycans intact.
TFMSA did lead to complete deglycosylation at 4 ºC, as can be observed in Figure 1B, lane 10, and did not give rise to protein degradation for BSA. On the
184 Dibenzoazacyclooctynes: Synthesis and Bioconjugation
other hand, applying the same procedure at room temperature resulted in loss of material for fetuin, although no protein degradation was observed for BSA.
From the condition screen, it was concluded that TFMSA was the best deglycosylation agent for our purposes, and further optimization of this reaction was performed. First, the optimal ratio between TFMSA and anisole was determined to be 4:1. Next, fetuin was treated with this mixture at different temperatures (-20 ºC, 4 ºC and room temperature) for different periods of time. A summary of this condition screen is shown in Figure 2. Lanes 2 and 5 show that no deglycosylation occurred at -20 ºC. On the other hand, deglycosylation at 4 ºC was complete within 1 hour (lane 3); treatment for 4 hours gave rise to only little backbone degradation, indicated by the small band below the band corresponding to deglycosylated fetuin (lane 6). Deglycosylation at room temperature was completed within 1 hour, with little protein degradation (lane 4); however prolonged reaction times gave complete degradation of the protein (lane 7).
Figure 2. SDS-PAGE analysis of fetuin deglycosylation using TFMSA/anisole (4/1) at different temperatures. (1) fetuin; (2) -20 ºC, 1 h; (3) 4 ºC, 1 h; (4) r.t., 1 h; (5) -20 ºC, 4 h; (6) 4 ºC, 4 h; (7) r.t., 4 h.
Notably, lanes 3 and 6 in Figure 2 seemed to indicate that after 4 hours more fetuin was obtained than after 1 hour. As both samples were obtained by taking an identical amount from the same reaction, the work-up procedure apparently did not give reproducible results.
Optimization of the work-up procedure showed that addition of three equivalents of a 60% pyridine solution is needed to neutralize TFMSA. Also addition was performed at -78 ºC as the reaction is highly exothermic. Further work-up was performed by dialysis of the protein against 250 mM NH4HCO3.
With the optimized procedure for fetuin in hand, the next aim was to test the same conditions for other glycoproteins. Therefore, one lightly glycosylated protein (lactoperoxidase) and one heavily glycosylated protein (alpha acid) were chosen. With this protein mixture a time screen was performed, using the same volume of TFMSA per mg of protein.
Figure 3 shows that lactoperoxidase is rapidly deglycosylated (lanes 1-4, Figure 3), with complete conversion after 1 hour (lane 2). Notably, the protein did not seem to degrade during prolonged exposure to TFMSA. On the other hand, alpha acid showed only poor deglycosylation after 1 hour (lane 6, Figure 3), indicated by the broad band in SDS-PAGE analysis. Also after 2 or 3 hours, fully deglycosylated alpha acid was not observed (lanes 7 and 8, respectively). Only after 4 hours, most of the alpha acid was fully deglycosylated (lane 9).
Figure 3. Deglycosylation of lactoperoxidase (lanes 1-4) and alpha acid (lanes 5-9) with TFMSA/anisole (4/1) at 4 ºC. (1) 0 h; (2) 1 h; (3) 2 h; (4); 3 h; (5) 0 h; (6) 1 h; (7) 2 h, (8) 3 h; (9) 4 h.
To investigate how the deglycosylation would proceed in a protein mixture, equal amounts of fetuin, alpha acid and lactoperoxidase were mixed, lyophilized and then treated with TFMSA for different amounts of time. The results of this reaction are shown in Figure 4. Lane 2 shows the mixture after 30 minutes, where only lactoperoxidase was fully deglycosylated, while alpha acid was still obtained with a broad mass range. After 1 hour (lane 3), only alpha acid remained partially deglycosylated, and after 3-4 hours all proteins were completely deglycosylated.
Remarkably, deglycosylation of alpha acid was now achieved faster than for the single protein experiment (Figure 3). This was probably because a larger amount of TFMSA was used compared to the amount of alpha acid (the volume of TFMSA was determined based on the amount of total protein).
In conclusion, the time required to reach full deglycosylation depends on the degree of glycosylation of a protein. Also, the amount of TFMSA/anisole used, compared to the amount of saccharides, seemed to influence the reaction time required. In protein mixtures a large variety of lightly and heavily glycosylated proteins is present, making it hard to determine a molar ratio between saccharide and TFMSA. By performing several experiments with different protein mixtures the amount of TFMSA/anisole mixture was determined to be optimal at 0.5 mL per mg of protein.
Figure 4. SDS-PAGE analysis of deglycosylation of a protein mixture of alpha acid, lactoperoxidase, and fetuin with TFMSA/anisole (4/1) at 4 ºC. (1) 0 h; (2) 30 min; (3) 1 h; (4) 2 h; (5) 3 h; (6) 4 h; (7) 5 h; (8) 6 h.
Having shown that deglycosylation can be achieved in a protein mixture using TFMSA at 4 ºC, the focus shifted towards cell lysates. To avoid loss of material after lysate deglycosylation, different work-up conditions were attempted on cell lysates.
As shown in Figure 5, dialysis against either 250 mM NH4HCO3 (which was used for the previous experiments) or Tris-buffer (20 mM Tris, 150 mM NaCl, 4% SDS)
186 Dibenzoazacyclooctynes: Synthesis and Bioconjugation
gave a reasonable recovery of cell lysate (lanes 2 and 3), whereas purification using Zeba Spin Desalting Columns27 against the same buffers gave no results (lanes 4 and 5). Alternatively, precipitation is a common purification method for cell lysates. Precipitation in acetone gave hardly any recovery (lane 6); on the other hand, precipitation in CHCl3/MeOH28 gave good lysate recovery in all mass ranges (lane 7).
Figure 5. Work-up conditions on cell lysate. (1) Cell lysate; (2) Dialysis with 250 mM NH4HCO3; (3)
Dialysis with 20 mM Tris, 150 mM NaCl, 4% SDS; (4) Zeba Gel Filtration against 250 mM NH4HCO3; (5) Zeba Gel Filtration against 20 mM Tris, 150 mM NaCl, 4% SDS; (6) Precipitation in
acetone; (7) Precipitation in CHCl3/MeOH.
As final test towards lysate deglycosylation, the procedure was performed in the presence of different salts. Up to now, proteins were always prepared by dissolving the commercially available glycoproteins in water, followed by lyophilization. However, cell lysates are commonly prepared in buffered solutions. To investigate the compatibility of the deglycosylation method with these buffer salts, fetuin was dissolved in different buffers including PBS, 250 mM NH4HCO3, Tris buffer (50 mM Tris, 1 mM β-mercaptoethanol, 0.32 M sucrose, 1 mM EDTA, pH 8), 6M Urea and HEPES-buffer (20 mM HEPES, 1% SDS, pH 8)), followed by lyophilization and TFMSA treatment.
Figure 6. Deglycosylation of fetuin in different cell lysate buffers, followed by lyophilization and treatment with TFMSA/anisole (4:1), for 4 hours. (1) Fetuin; (2) PBS; (3) 250 mM NH4HCO3; (3)
Tris-buffer; (5) 6M Urea; (6) HEPES-buffer, pH 7.9; (7) MilliQ.
Results of this experiment are shown in Figure 6, which shows that almost all buffers are compatible with TFMSA, resulting in full deglycosylation after 4 hours. 6M Urea (lane 6) is not suitable for deglycosylation because after 4 hours deglycosylation was still incomplete.
In conclusion, optimal deglycosylation without significant protein degradation was achieved using the following reaction conditions: a mixture of TFMSA and anisole (ratio 4:1) at 4 ºC for 4 to 6 hours with 0.5 mL per mg of protein. The reaction was quenched with 3 volumes of 60% pyridine and extracted with Et2O, followed by dialysis of the aqueous phase against 250 mM NH4HCO3 for simple protein mixtures, or precipitation in CHCl3/MeOH for cell lysates.
7.3 Cell Labeling studies
To obtain as many glycoproteins as possible after enrichment, it is important that glycan labeling is a highly efficient process. In general, live cell labeling studies have looked at the overall amount of labeling obtained, instead of at the range of proteins labeled, or at which cellular compartments contain the labeled glycoproteins.17, 29, 30 To investigate which conditions are best to label the largest range of glycoproteins, whether only the cell membrane is labeled or also other cellular compartments,31 and which azide-reactive probe would be best for introduction of an enrichment probe, we introduced GalNAz to the glycoproteins of Jurkat cells and subsequently labeled the azides with a biotin, using either the Staudinger ligation of SPAAC, shown in Scheme 3. In our optimization efforts we applied a wide range of cyclooctynes and one Staudinger reagent. Also, the concentration of reagent and reaction time were optimized.
Scheme 3. Schematic representation of cell surface labeling using SPAAC, with biotinylated