The FASP FACE method was proven to effectively enrich glycopeptides as observed by the TIC profile, figure 57. However, since the method uses ultra-centrifugal filters there is the possibility that some glycopeptides are lost in the washed in the step to remove non glycopeptides, that some remain bound to the lectin or that some are bound to the filter itself during both the FASP and the FACE stage. Amicon state that their 30 kDa ultra centrifugal filter has a 95% recovery of BSA when 4 mL is concentrated to 0.2 mL. However, the numerous washes involved in FASP FACE means that this ideal 95% would be lower. There are also proteins of lower Mw present in the sample which are more likely to be lost after many centrifguations. A E E K Pyroglutamic acid? GlcNAc Homoserine lactone? F?
In an effort to reduce the loss of glycopeptides, thereby improving the quality of the sample and subsequent mass spectrometry, magnetic beads bound to streptavidin were investigated. In theory, these beads can be bound to any lectin conjugated to biotin and be used in a pull downs of glycopeptides. The can be boiled to more efficiently release the bound peptides from the lectin and the number of washing steps is drastically reduced, figure 59.
Figure 59 – Use of lectin bound to beads for enrichment of glycoproteins
Since the presence of P. falciparum N-glycoproteins is not yet proven, initial attempts at using magnetic beads were with T. brucei lysates and the lectin ricin due to the high abundance of Gal terminal N-glycans. T. brucei lysates were incubated with biotinylated ricin before MACs streptavidin beads were added. The beads were washed and bound proteins eluted by adding boiling laemmli buffer and loaded on to an SDS gel, gel A figure 60. The amount of protein loaded into lane 2 was a quarter of that used in the enrichment experiment. The enrichment seems to have been successful as a number of proteins appear higher in abundance in the enriched compared to the non. The same assay was therefore applied to P. falciparum lsyates.
A B
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Figure 60 – A: Magnetic bead-bound ricin enrichment of T. brucei. Lane 1 = ladder, lane 2 = T. brucei lysates and lane 3 = T. brucei proteins eluted from the magnetic bead-bound ricin. B: Magnetic bead- bound GSLII and WGA enrichment of P. falciparum. Lane 1 = ladder, lane 2 = P. falciparum lysates, lane 3 = blank, lane 4 = P. falciparum proteins eluted from the magnetic bead-bound GSL II and WGA,
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This time GSL II (lane 5), WGA (lane 6) or both lectins (lane 4) were used to enrich the potential short GlcNAcs-proteins, gel B figure 60. Unfortunately, no proteins were seen in any elution lane (4 to 6) and the lane containing around a quarter of the proteins compared to that used in the enrichement assay only showed a poor smear with barely any protein present. There was also a need for a positive control – a protein that contains a GlcNAc for certain. BSA-GlcNAc is a commercially available protein that has been chemically labelled with a GlcNAc and is sold as a control in agarose-lectin binding assays of WGA or GSL II. It is can therefore be used as a positive control in our magnetic bead-lectin assay to determine if the lectins are not binding to P. falciparum lysates due to the absence of GlcNAc or if there is a problem with the lectin. Furthermore, binding the lectin to the proteins and then to the beads could lead to lectin that is not bound to any protein binding to the bead and diluting the result. Therefore, binding the lectins to the beads first was tested with the BSA-GlcNAc control. The beads were washed before the addition of the protein so that any excess lectin was removed. Both lectins were again tested together and separately, gel A figure 61. Only some high molecular weight oligomers of BSA-GlcNAc are seen in elution lanes 4 to 6. Four times more BSA-GlcNAc was used in the enrichment experiment compared to the control lane 2, therefore the small amount of high molecular weight proteins is nothing in comparison. From this experiment, it is also not possible to tell if the binding of the oligomers is due to the GlcNAc or non-specific interactions. A negative control should have been used where the lectin is not present. The band of protein at the bottom of the gel in lanes 4 to 6 is from streptavidin. It is possible that more lectin is needed than was used. The beads have a high enough loading of streptavidin to conjugated around 20 x more lectin than was used in this assay. Using more lectin could improve the binding of BSA-GlcNAc as steric blocking of the few lectins conjugated could have prevented more proteins from binding, especially as it seems like the predominant form of the protein is as oligomers.
A B
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Figure 61 - A: Magnetic bead-bound GSLII and WGA enrichment of BSA-GlcNAc. Lane 1 = ladder, lane 2 = BSA-GlcNAc, lane 3 = blank, lane 4 BSA-GlcNAc eluted from magnetic bead-bound GSL II and WGA, lane 5 = BSA-GlcNAc proteins eluted from magnetic bead-bound GSL II and lane 6 = BSA- GlcNAc proteins eluted from magnetic bead-bound WGA. - B: Magnetic bead-bound GSLII and WGA enrichment of BSA-GlcNAc and P. falciparum. Lane 1 = ladder, lane 2 = BSA-GlcNAc, lane 3 = P. falciparum lysates, lane 4 BSA-GlcNAc eluted from magnetic bead-bound GSL II and WGA, lane 5 = BSA-GlcNAc proteins eluted from magnetic beads without lectin, lane 6 = P. falciparum proteins eluted from the magnetic bead-bound GSL II and WGA and lane 7 = P. falciparum proteins eluted from magnetic beads without lectin.
The assay was repeated with 10 x more lectin with both the positive control BSA-GlcNAc and fresh P. falciparum lysates and the negative control of beads that were not conjugated to lectins and freshly purchased Dynaeads. There was only a slight increase in BSA-GlcNAc in lane 4 where lectins were present compared to lane 5 where they are absent. This means that the interactions of the BSA-GlcNAc oligmoers is predominantly non-specfic. It could be that the high density of proteins in the oligomer sterically hinders the GlcNAc residue from binding. However, since this is sold as a standard for GSL II and WGA it would be unusual. There are no
P. falciparum proteins present in lane 6 where lectin was present nor lane 7 where it was
absent. In a way, this is a positive result since it means that the proteins do not interact with the beads in a non-specific manner. However, no glycoproteins were enriched. The band in lanes 4 and 6 just above 25 Da is biotin. GSL II has a molecular weight of 113 kDa and WGA 38 kDa. The action of boiling in laemmli buffer breaks the streptavidin bond releasing the lectins from the beads, but there are no bands of this Mw seen on the gel. Both lectins exist as dimers and although they were subjected to reducing conditions it is possible that they have remained as dimers (GSL II at 226 kDa, WGA at 76 kDa). There are bands at these molecular
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the lectins. It could be that the lectins needed longer than what they were given to react with the dynabeads.