Because sample labeling and cleanup is laborious and time consuming, direct label-free detection and identification by MS
is a popular technique. MS also has the advantage of being able to provide exact mass data and identify very complex glycan
profiles. Matrix-assisted laser desorption time-of-flight (MALDI–TOF) MS is one of the MS techniques often used. However,
because MALDI–TOF-MS is not coupled to chromatographic separation, isomeric information is not obtained. Sample preparation
for MALDI–TOF-MS involves deglycosylation, purification, acidification, and cation exchange to remove salts after deglycosylation,
and takes about 6 h (see Figure 1) (7).
Figure 1: Characterization workflows vary widely in terms of the techniques used and the time required to perform them. Even
the fastest workflow is not fast enough to be practical for real-time process monitoring. LC-LIF is laser-induced fluorescence.
LC is liquid chromatography. CE–MS is capillary electrophoresis–mass spectrometry. HPLC is high-performance liquid chromatography.
MALDI is matrix-assisted laser desorption. (ALL FIGURES BUT FIGURE 3 ARE COURTESY AGILENT TECHNOLOGIES.)
Hyphenated techniques have been developed to draw upon the combined attributes that labeling, chromatographic separation,
and MS provide: improved detection sensitivity, isomer identification, and accurate mass information. For example, CE–MS methods
enable faster separations as well as separation of isomeric species (8). HILIC and reverse-phase HPLC methods can be used
in combination with electrospray MS detection. These methods also provide useful isomeric information (9, 10). Though these
hyphenated methods provide powerful data, they often require 2–3 days to complete, thereby making them unsuitable for real-time
Because porous graphitized carbon (PGC) chromatography is amenable to the separation of unlabeled glycans, it, in combination
with nanoelectrospray MS, is emerging as the preferred hyphenated technique to separate and identify glycans without derivitization
(11, 12). Unlike reverse-phase HPLC, glycans are readily retained on PGC without the need for a labeling step. However with
this method, the glycans must be reduced to simplify their chromatographic profile. Without this lengthy reduction step, each
glycan yields two peaks due to the anomeric carbon at the reducing end.