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In order to perform an effective glycomic analysis, glycans have to be released from their glycoconjugates. As mentioned earlier, glycoproteins contain two types of glycans, that is, N-glycans and O-glycans. All N-glycan structures can be digested from proteins by peptide:N-glycanase F (PNGase F) except for 1.3 core fucosylated N-glycans which are commonly found in plants and invertebrates. They can be removed by peptide; N-glycanase A (PNGase A) (Tretter et al. 1991). In addition, it has recently been suggested that the two newly cloned FuTIO and FuTl 1 can catalyse Î±l,3-fucosylation to N-glycan core in humans (Mollicone et al, 2009).
In contrast, there is no enzyme that can digest all O-linked glycans efficiently. In this case, chemical release is the only practical option. An example is reductive elimination, also known as alkaline P-elimination which was first introduced in 1966 in an analysis of oligosaccharides from porcine submaxillary mucins (Carlson, 1966). At present, this is the most common method used to release O-glycans from proteins.
Reductive elimination is a technique whereby unwanted degradation or 'peeling' of the oligosaccharides is avoided by reducing the monosaccharide of the reducing terminal to an alditol using reducing agents such as potassium borohydride (KBH4) in an alkaline solution such as potassium hydroxide (KOH) (Carlson, 1966). During the chemical degradation, hydroxide from the alkali attacks the hydrogen on the backbone of the glycan-linked amino acid leading to the elimination of the O-linked sugar as illustrated in Figure 2.5. The reducing terminal glycan (O-GalNAc) is then reduced, thus breaking the ring structure. Reductive elimination can be performed either before or after digestion of N-glycans, but the latter usually results in some contamination with reduced and non-reduced N-glycans, especially when high mannose N-glycans are present.
Figure 2.5 Reaction steps for reductive elimination of O-linked glycans. R, 3-linked saccharide; R1, 6-linked saccharide
2.5.2 Enzymatic and Chemical Digestions of Glycans
The enzymatic and chemical digestions of glycans are an essential part of glycan screening and sequencing because they can provide information on structural features such as stereochemistry and linkage (Dell et al, 2007). Enzymatic digestions are normally for stereochemistry and/or linkage specific but must be carried out on native glycans. For example, Î²1,4-galactosidase digestion is used to determine the type of terminal Gal population in the sample of interest. In contrast, chemical digestions are used to degrade sugar residues with broader specificities and can be carried out on either native or permethylated glycans. For example, trifluoroacetic acid (TFA) degradation at room temperature is used to remove terminal fucose residues. In addition, time course degradation is possible because certain fucose linkages are more labile. Thus, 3- and 4-fiicose residues are more likely to be released first than 2-and 6-fucose which allows easy confirmation of fucose and rough determination of their linkages. Mass spectrometry is an ideal method to monitor the products of these reactions because of its sensitivity.
2.5.3. Enzymatic Release for Glycan Analysis
Endoglycosidases and glycoamidases are usually used to release N-glycans from glycoproteins as illustrated in Figure 2.6. Peptide N-glycosidase F (PNGase F) is widely used for the intact N-glycan release, because it allows the release of almost all types of N-glycans. However, P-NGase F is unable to cleave N-glycans which have Î±1-3 fucosylated residue attached at the reducing-terminal GlcNAc. This substitution is commonly found in insect and plant glycoproteins. In this case, PNGase A is used to release Î±1-3 core fucosylated N-glycans (O'Neill et al., 1996).
In contrast to N-glycans, there is no universal enzyme capable of releasing all O-glycans as only endo-Î±-Nacetylgalactosaminidase can release unsubstituted Core 1 type O-glycans (Pochec et al., 2003). Consequently, chemical release methods are universally used to cleave O-glycans (Mechref and Novotny, 2002).
Figure 2.6 Glycosidase sites of PNGase F, endoglycosidase, and exoglycosidase with disialylated biantennary, core-fucosylated N-glycan (Wells et al., 2001; ) Rexach et al., 2008).
2.5.4 Chemical Derivatization for Glycan Analysis
Permethylation is a common derivatization method used for N- glycans or O-glycans and has been used for many years in the analysis of carbohydrate. In this regard, two derivatization methods have been widely used for permethylation, as reported by Hakomori (Spiro, 2002) and Ciucanu and Kerek (de Beer et al., 1995). However, currently, Ciucanu and Kerek's procedure is more commonly used for permethylation of glycans. It utilizes iodomethane, dimethyl sulfoxide and sodium hydroxide as illustrated in Figure 2.7. This popular permethylation method has been further modified through the adaptation of solid-phase chemical derivatization (Voet, 2004).The modified procedure is better because it presents rapid reaction time, cleaner reaction products and is a simple procedure.
Permethylation presents several advantages for the mass spectrometry (MS) analysis of glycans. Firstly, permethylated derivatives makes it easy to determine glycan structural type, for example, branching and interglycosidic linkage information. Secondly, permethylation stabilizes the sialic acid residues in acidic glycans resulting in more predictable ion products because sialic acids are unstable and easily decomposed during fragmentation. In addition, permethylation neutralizes sialic acids thereby allowing a simultaneous analysis of neutral and acidic glycans in the positive ion mode. This will allow the comparison of natural abundances of both neutral and acidic glycans. Last but not least, permethylated glycans ionize more efficiently than the native glycans during an MS analysis. Thus as Mehta et al., (1996); and Viseux et al., (1996) noted,
Permethylated glycans provide very detailed structural information especially when used with electrospray ionization (ESI) MS, MALDI ionization, and collision-induced dissociation (CID
Figure 2.7 Schematic illustration of permethylation.
2.6 Glycan Fragmentation
In tandem mass spectrometry, sample molecular ions are activated and fragmented in order to provide detailed structural information. The MS2 experiment is the most common type of analysis and can be done using most modern mass spectrometric instruments. However, multiple stage mass spectrometric analysis (MSn), especially MS3, is fast gaining recognition as a more effective method for glycomic and glycoproteomic analyses due to the increasing demand for detail structural information. Furthermore, MSn data could indicate which fragmentation pathway the MS2 fragment originated from, which is particularly useful for analysis on complex glycan structures (Harvey, 2009). It should be noted that most of the current knowledge on glycan fragmentations is derived from the analysis of oligosaccharides using FAB-MS instruments during the 1980's, for example, the work of Dell & Ballou (Dell & Ballou, 1983).
The fragmentation pathways of glycans in tandem mass spectrometric analysis are varied. In contrast to native samples, permethylated glycans fragment in a very predictable way in both positive and negative ion modes. On the whole, the positive ion mode is the method of choice due to the better fragmentations most commonly observed with singly charged sodiated ([M+Na]+) and protonated ([M+H]+) molecular ions in MALDI, or as multiply charged ions in ESI. On the other hand, in the negative ion mode, the molecular ion is deprotonated, making this mode of ionisation best suited to molecules that have an intrinsic negative charge, for example, sulphated or sialylated glycans, on condition the latter are not permethylated. The negative mode is also very suitable for the analysis of underivatised glycans on condition the resulting molecular ions are stable during analysis. But this is often not the case in MALDI where sulphates and sialic acids are readily lost. In contrast, ESI is very suitable for handling fragile molecular ions. As an example, Karlsson et al. have very successfully performed rigorous characterization of neutral and underivatised O-glycans reductively eliminated from human MUC5B using LC-ESI-MSn (Karlsson et al, 2004).
In MALDI and ESI instruments, various types of cleavages can be observed in CED analyses of permethylated glycans, for example, in the fragmentation of a sialylated core 2 O-glycan as illustrated in Figure? The most common type of cleavage is the glycosidic cleavage with hydrogen transfer, also known as p-cleavage, with charge residing on either end (see Figure?). This type of cleavage can occur in both positive and negative modes. An important cleavage normally accompanying glycosidic fragmentation with hydrogen transfer is p-elimination of the substituent from position 3 of a HexNAc residue (Dell, 1987; Dell et al, 2008) (Figure)? These glycosidic and P-eliminated fragment ions can provide information on sites of sialylation, fucosylation and branching as well as the number of repeatations of LacNAc.
Another type of fragmentation commonly observed in oligosaccharide analysis is ring cleavage (cross-ring fragmentation) as illustrated in (Figure 1.20). where is the figure? Cross-ring fragmentation is prominent in multiple stage MS (MS3 and higher) but it can also occur in MS2 experiments provided it is carried out in a high-energy collisional activation. Sodiated molecular ions produce better ring-cleavages compared with their protonated counterparts (Dell & Ballou, 1983; Dell, 1987; Zaia, 2004). In a cross-ring fragmentation, the charge can remain on either the reducing or the non-reducing end, depending on the nature of the sample and the ionisation mode. This type of fragmentation is especially useful for assigning linkages.
In other mass spectrometric techniques, especially in FAB instruments, permethylated derivatives very commonly undergo A-type cleavages on the non-reducing side of a glycosidi bond to give oxonium-type fragment ions, which may carry sodium if the parent ion is sodiatec This A-type cleavage is often favoured at HexNAc residues and can only occur in the positive ion mode. Secondary cleavage Î²-elimination of the substituent from the position 3 of a HexNAc residue is often observed as well (Dell, 1987; Dell et al., 2007). It should be noted that the cleavage of sialic acid residues often leads to the formation of oxonium ions even in MALDI and ESI instruments as illustrated in Figure ?