Analysis of Released Glycans
Cleanup of Glycan Samples
Although MALDI and, to a lesser extent, electrospray ionization (ESI) are comparatively tolerant to the presence of contaminants, it is important to desalt samples before analysis in order to produce acceptable spectra. Suitable methods include the use of resins , aminopropyl silica in a hydrophilic interaction liquid chromatography (HILIC) microelution plate , or gra- phitized carbon . Resins are usually packed into microcolumns or can be added directly to the MALDI target . Alternatively, membranes such as low molecular cutoff dialysis membranes or Nafion 117  can be used. Peptidic material can be removed from glycan samples with C18 such as that incorporated into ZipTips. Among other cleanup methods is the use of ZIC- HILIC and cotton wool  and oxylamino-containing polymers to bind the reducing terminus of the glycan followed by release with mild acid .
Derivatization at the Reducing Terminus
Because glycans do not fluoresce or absorb satisfactorily at UV frequencies, derivatization of the reducing terminus by addition of fluorescent tags such as 2-aminobenzamide (2-AB) , 2-aminobenzoic acid (2-AA) [113-115], or 2-aminopyridine (2-AP) [116, 117], usually by reductive amination (Figure 3.3), is routine for the detection of glycans. Such derivatives have also found use in mass spectral analyses for enhancement of signals or for modification of fragmentation patterns.
Because of the presence of amine groups linking the derivative to the glycan, these derivatives can increase the proton affinity of the molecules when [M+H]+ ions are formed in preference to the more usual [M+Na]+ ions. With ESI, for example, they have been reported to enhance signal strength by 30- to 100-fold depending on the derivative . Recently procainamide (A-(2-diethylamino)ethyl-4-aminobenzamide), first introduced in 2000 , has received considerable interest in this context and is available in a kit from ProZyme. Derivatization with aminobenzoic acid alkyl esters has been used to increase hydrophobicity, which results in an increase in sensitivity [120, 121], particularly for fast atom bombardment (FAB) ionization. Considerable increases in sensitivity have also been achieved by using derivatives containing a constitutive cationic charge such as those prepared using trimethyl(4- aminophenyl)ammonium chloride (TMAPA)  or Girard's reagent T . Derivatives containing a bromine atom have been used as labeling reagents because of the distinctive bromine isotope pattern that allows all fragment ions containing the derivatized end of the molecule to be identified [123, 124].
Figure 3.3 Reductive amination reaction for derivatization of the reducing terminus of carbohydrates (shown for the chitobiose core of N-glycans) and some of the typical amines used for the reaction.
The reductive amination reaction with 2-AP can be reversed to recover the carbohydrate . Kuster et al.  have studied the effect of a number of derivatives on the high-energy fragmentation of N-glycans; more cross-ring fragments were seen than with underivatized compounds. An 0,2A-ion (see following text) from the reducing terminus (loss of the derivative) was often the most abundant ion in the spectrum.
An alternative derivatization strategy that avoids some of the problematic cleanup procedures necessitated by reductive amination includes the production of hydrazone and hydrazide derivatives as illustrated by reactions with phenylhydrazine [127-129]. An advantage of this reagent is that reactions are performed under slightly basic conditions causing minimal loss of sialic acid. Also, the derivatives form prominent [M+H]+ ions, making them derivatives suitable for the detection of glycopeptides in the presence of peptides . Thus the glycan profile and site occupancy (Asn-to-Asp conversion) could be monitored in a single spectrum. Basic and quaternary ammonium-containing derivatives linked via hydrazine chemistry have also been used for this purpose .
Also of interest are the derivatives containing a cationic charge [122, 131, 132]. Of these, Girard's T reagent (carboxymethyltrimethylammonium chloride hydrazide) [122, 132] has received the most interest and has been used for quantitative studies of N-glycans in positive ion mode because it eliminates the differences in ionization efficiency inherent in the production of [M+H]+ and [M+Na]+ ions [133, 134].
Because PNGase F releases the glycans as the glycosylamines, it is possible to prepare derivatives directly by reaction with carbonyl reagents. Chen and Novotny  have prepared such derivatives from 2-methyl-3-oxo-4- phenyl-2,3-dihydrofuran-2-yl acetate immediately after release.
A large number of other reducing-terminal derivatives have been investigated and are reported in recent reviews [136-138].