molecule as a result of which our sialic acid can show fluorescent character, and can be detected by means of a fluorescence detector. It is vital to notice that the OPD reagent prepared was close to 11.9 mg/mL because a slight increase in concentration would have severely affected the solubility of OPD in the mixture of RODI water and sodium bisulfate. Moreover, as a result of the relative hydrophobicity of the OPD labeled NANA, it was also vital to use reversed phase HPLC as compared to other kinds of HPLC techniques. The use of mobile phase A and B were determined as a result of a reference which performed a similar experiment [1]. The prominent reasons for failure were addressed to develop a more efficient technique to suit our lab. The analysis of the sialic acid content in different batches of our fusion glycoprotein product was relative. It can accurately indicate if some batches were hyper-sialated or hypo-sialated. The exact content of the sialic acid in the glycoprotein cannot be determined through the experiment. The molar mass of our fusion glycoprotein product was estimated to be about 62.5 kDa. The results obtained are intended to be verified through mass spectrometry. This would be done over the course of next semester. One critical observation made during the experiment was regarding the discrepancy of the NANA to protein ratio for the 10008 batch. It was observed that the mole ratio went from 2.13 to 4.10 for the batch. The principle error can be attributed to lack of precision over pipetting during sample preparation. The error would be addressed over the course of next semester. Since, the Graph 2 obtained for trial 2 is not clear, it becomes vital to redo the triplet injections before proceeding with future work. Analyzing Graph 2, it becomes clear that the peak area for 25 ng/mL NANA standard seems inaccurate relative to Graph 1. This might also be attributed to imprecise pipetting. These problems should get addressed when a trial 3 of triplet injections is repeated. It is also thought that there might be a problem within the HPLC which might not let exactly 13% mobile phase B to be delivered. As a result, an isocratic method would be performed for trial 3. For this trial, 87% mobile phase A and 13% B would be prepared individually, and would 62 UMLJUR
be mixed before the experiment. Then, the HPLC would be told to run 100% new solvent A prepared. This might answer a few problems as well. One other problem that has been looming over the entire course of the experiment is the detection of other peaks before the appearance of the OPD labeled NANA peak. This might be because of some other kinds of sialic acid that might be present in our standard NANA since it is not 100% pure. In fact, the presence of NAGA is highly probable. Hopefully, it will become clear after the analysis through mass spectrometry what those peaks actually indicate.
CONCLUSION
A few conclusions have been reached from the research carried out over the course of last semester. Firstly, it can be concluded that a successful experimental procedure for the detection of sialic acid was devised. Also, successful demonstration of the relative NANA content within our fusion glycoprotein product was performed. Moreover, the results from both trials show reasonable reproducibility. However, it will be critical to try to reproduce the data once again, since for the sample of 10008 Lot, there is a discrepancy.
RECOMMENDATIONS
There are many aspects of this research which must be examined in future studies. First of all, it is critical to reproduce the results as soon as possible. It is vital to establish a consistency of the glycan content within the protein due to its effects on protein structure and function. As discussed, it is also critical to analyze the chromatograms with a mass spectrometer. This can not only verify our results, but can also possibly convey the reason for the detection of other peaks in the chromatograms. Lastly, it would be interesting to cleave the intact glycans from the linkage between the protein and carbohydrate chain from the fusion glycoprotein product. The effect of this on protein structure and function can also be studied. 1.Anumula, K .R. Rapid Quantitative Detection of Sialic Acids in Glycoproteins by High Performance Liquid Chromatography with a Sensitive Fluorescence Detection. Anal. Biochem., 1995, 230, 24-30.