A wide variety of glycans exist in mammalian cells with a wide variety of functions. They are involved in cell structure, the immune response, and cell-cell communication to name but a few. N-glycans are a particularly abundant form, and thus, studying them can provide a lot of useful information.
Abnormal glycan structure and glycosylation are associated with a range of diseases, including immune deficiencies, cardiovascular disease, and cancer.1 The study of glycan structure is therefore very important in understanding such diseases. However, as glycans vary greatly and commonly exist at very low concentrations, there are often difficulties when analyzing their fine structure. New techniques are being developed to combat these setbacks and to aid in monitoring, diagnosis, and prognosis of disease. Gaining insight at a molecular level could also aid in drug and cure development.
Analyzing glycan structure is one essential component of understanding glycosylation patterns, and mass spectrometry is used to do this. Variation in glycan structures is due to the presence of N-acetlylated and acidic residues, which can affect the capability of glycans to be ionized.2 Therefore, permethylation of glycans is often performed before mass spectrometry. This involves the reaction of glycans with methyl iodide in the presence of sodium hydroxide beads, which cleave glycosidic linkages. This reaction stabilizes the acidic residues in glycans, meaning they ionize more uniformly and efficiently due to lower variation in their chemical properties.2,3 Traditional permethylation procedures, however, can produce side reactions. The high pH of sodium hydroxide can lead to the destruction of glycans through oxidative degradation, as well as peeling reactions.3 Moreover, as glycans exist at very low concentrations (low picomole to femtomole), these side reactions can become adversely prominent, meaning the ability to permethylate trace levels of glycans is significantly reduced.
Thus, a miniaturized version of common solid-phase permethylation techniques was used by Desantos-Garcia and colleagues4 to study N-glycans. There are various classes of glycans produced from glycosylation, which differ from the amino acids that they link to on the polypeptide chain. N-glycans are the most common class of glycans and are attached to a nitrogen atom of asparagine or arginine. The technique used by Desantos-Garcia and colleagues4 involves the packing of sodium hydroxide beads in a microcolumn format, thus reducing the amount of sodium hydroxide present at the end of permethylation and minimizing oxidative degradation and peeling reactions. Desantos-Garcia and colleagues4 also found that reducing the amount of sodium hydroxide beads did not significantly diminish the permethylation efficiency, provided enough methyl iodide was used.
After solid-phase permethylation, N-glycans were on-line-purified using a common LC-MS setup.4 The most common methods of purification are liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Both of these methods, however, are time consuming and involve numerous purification steps. Moreover, LLE risks the efficiency of sample recovery, and SPE has limited throughput. Therefore, using on-line purification prior to LC-MS means sample handling and transfer steps are reduced, thus reducing the surface area that a sample comes into contact with, minimizing sample losses and enhancing analytical sensitivity. When compared to LLE and SPE, on-line SP purification yielded more intense mass spectrometry signals for permethylated N-glycans, at 75% and 95% higher levels than for LLE and SPE, respectively.4 This on-line purification with reduced sample handling was possible due to the reduced sodium hydroxide in samples, the result of miniaturized solid-phase permethylation.
Thus, the use of miniaturized solid-phase permethylation to reduce the amount of sodium hydroxide, in conjunction with on-line purification, allowed enhanced mass spectrometry sensitivity to N-glycan structures. This is vitally important in determining fine structural differences between glycans, which in turn is needed to better understand glycosylation in both normal and abnormal cases. The more we understand about the glycosylation process, the closer we get to understanding, diagnosing, and curing diseases such as heart disease, immune deficiency, and cancer.
1. Dennis, J.W., Granovsky, M., and Warren, C.E. (1999) ‘Protein glycosylation in development and disease‘, Bioassays, 21 (5), (pp. 412-421)
2. Alvarez-Manilla, G., et al. (2007) ‘Tools for glycomics: Relative quantitation of glycans by isotopic permethylation using 13CH3I‘, Glycobiology, 17 (7), (pp. 677-687)
3. Kang, P., et al. (2005) ‘Solid-phase permethylation of glycans for mass spectrometric analysis‘, Rapid Communications in Mass Spectrometry, 19 (23), (pp. 3421-3428)
4. Desantos-Garcia, J.L., et al. (2011) ‘Enhanced sensitivity of LC-MS analysis of permethylated N-glycans through online purification‘, Electrophoresis, 32 (24), (pp. 3516-3525)