Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Matrix-Assisted Laser Desorption/Ionization (MALDI)
The use of MALDI to ionize carbohydrates was first reported by Karas and Hillenkamp in one of their early papers  and was applied to N-linked glycans by Mock et al. in 1991 . It has since become one of the most popular techniques for glycan analysis, mainly because it tends to produce only one ion from carbohydrates, thus providing a means to easily profile glycan mixtures (for reviews, see [11, 198-204]. Many matrices have been developed (see Table 3.2 for examples), but one of the earliest, DHB , is still the most popular.
Although the dried droplet method of sample preparation works reasonably well, targets prepared in this way with DHB have large crystals pointing from the periphery toward the center. Studies by Kussmann et al.  have suggested that there, at least for peptides, is considerable fractionation of cations between the crystals and noncrystalline areas, giving very different signals from different areas of the target. More homogeneous targets and, consequently, better performance have been achieved by techniques such as mixing the DHB with various additives including 2-hydroxy-5-methoxybenzoic acid  (the mixture is commonly known as super-DHB), 1-aminoisoquinoline
Table 3.2 Matrices for MALDI mass spectrometry of carbohydrates.
Table 3.2 (Continued)
Table 3.2 (Continued)
(HIQ) , fucose , or spermine . Recrystallization of the initially dried sample spot from ethanol  is also popular in producing a more homogeneous target surface and better mixing of sample and matrix. Strong signals that rapidly fade on firing the laser at a particular sample spot suggest that the carbohydrates coat the surface of the target crystals rather than being incorporated into the crystal, as is thought to be the case with peptides. Typical MALDI-TOF spectra of N-glycans are shown in Figure 3.4a.
Figure 3.4 (a) Positive ion reflectron MALDI-TOF spectrum of N-linked glycans released with hydrazine from chicken ovalbumin. Major ions are [M+Na]+. (b) Positive ion reflectron MALDI-TOF spectrum of a disialylated biantennary N-linked glycan showing loss of sialic acid residues to give focused ions and PSD ions (marked with an asterisk).
Unlike neutral glycans, sialylated glycans are relatively unstable under MALDI conditions and readily eliminate sialic acid. Operation of MALDI- TOF instruments in linear mode restricts the observation of fragmentation occurring in the flight tube and is preferred by some investigators . In reflectron mode, such decompositions within the instrument produce broad metastable ions (Figure 3.4b) whose mass can be predicted by the formula
where Mc is the mass of the metastable ion, Ma is the mass of the parent ion, Mb is the mass of the fragment, and r is a constant that is dependent on the instrument . Migration of the labile acidic proton of the carboxy group is responsible for sialic acid loss, but this can be prevented by derivatization such as methyl ester formation  (Figure 3.5a), permethylation , conversion to amides [216, 217], or ion pairing with quaternary ammonium or phosphonium salts . These techniques not only stabilize the sialic acids but also prevent negative ion and salt formation, thus allowing quantitative glycan profiling to be made in the positive ion mode. Methyl ester formation of sialic acids with methanol catalyzed by 4-(4,6-dimethoxy-1,3,5-triazin-2- yl)-4-methylmorpholinium chloride (DMT-MM) is particularly useful because a2 ^ 6-linked sialic acids form methyl esters, whereas a2 ^ 3-linked sialic acids form lactones (Figure 3.5b). The 32 mass unit difference is easily detectable by MS and provides a quick method for determination of sialic acid linkages .
Use of different matrices can also be used to reduce sialic acid loss. Thus, 2,4,6-trihydroxyacetophenone (THAP) has been reported to cause less sialic acid loss, particularly when mixed with ammonium citrate , and ATT has also shown promise . With a standard nitrogen laser, “softness” of the matrix is roughly in the order CHCA » DHB > sinapinic acid (SA) ~ THAP > ATT > hydroxypicolinic acid (HPA) . Infrared lasers with glycerol or 3- nitrobenzyl alcohol matrices can also be used to reduce sialic acid loss [222-224] but are generally not found on commercial instruments. Ice has also been used as the matrix by use of a Peltier-cooled sample stage to provide conditions approaching those found physiologically . Atmospheric pressure ion sources have also found application in this context [226, 227].
Figure 3.5 (a) Released glycans from bovine fetuin derivatized as methyl esters with methyl iodide. (b) The same glycans derivatized with methanol in the presence of DMT-MM. a2 ^ 6-linked sialic acids form methyl esters, whereas a2 ^ 3-linked acids produce lactones. The mass difference between these species allows the linkage to be determined.
Sulfated glycans are also unstable under MALDI conditions and eliminate sulfate easily . Their sodium salts, however, are more stable [97, 229] and can be differentiated from phosphorylated glycans (phosphate and sulfate have the same nominal mass) by the observation that whereas phosphorylated glycans ionize as free acids, sulfated glycans are invariably seen in the positive ion spectra only as sodium salts . In order to stabilize sulfates, ion pairing with the tripeptide Lys-Lys-Lys has been used  with the added advantage that it enables isobaric phosphates and sulfates to be differentiated by their fragmentation spectra . Whereas the sulfates showed preferential cleavage of the oxygen-sulfur bond, the phosphates preferred to eliminate the ligand. Fragmentation has also helped in the location of phosphate groups on high-mannose glycans; whereas 6-linked phosphates are stable, 1-linked phosphates undergo cleavage in their PSD spectra . Derivatization has also been used to examine sulfated glycans . Samples were first permethylated and then subjected to methanolytic cleavage of the sulfate groups (which do not methylate) to reveal the linking hydroxyl group. The desulfated permethyl- ated glycans were then subjected to further permethylation using deuterome- thyl iodide to label the newly exposed hydroxyl groups.
In positive ion mode, MALDI produces mainly [M+Na]+ ions from neutral glycans [197, 198, 234] (Figure 3.4a) although other cations can be introduced by doping the matrix with an appropriate salt. Sialylated glycans produce a mixture of [M+Na]+ and [М-иИ+(и + 1)Na]+ ions in positive mode and [M-H]- ions under negative ion conditions. Negative ions of neutral carbohydrates have only been reported occasionally from specific matrices such as p-carbolines [235, 236]. However, doping the matrix with salts, particularly chlorides, has yielded abundant [M+adduct]- ions. THAP doped with ammonium nitrate , harmine doped with ammonium chloride , and 9-aminoacridine doped with sodium iodide or ammonium chloride  are recent examples.
MALDI is mistakenly not generally regarded as a quantitative technique. However, if experiments are performed appropriately, good quantitative data can be produced. It is important that target inhomogeneities are overcome by acquiring spectra with many laser shots directed to several target positions . One method for reducing target inhomogeneities is to use an ionic liquid matrix  such as that prepared from a standard MALDI matrix and an organic base such as butylamine . The abundance of the [M+Na]+ ions normally formed by carbohydrates appears to show little variation with N-glycan structure [211, 234, 242, 243] unlike the case with peptides where ionization by protonation reflects the proton affinity of the constituent amino acids.
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