The choice of technique depends the amount of information required. Although the first, more conventional method of releasing
the glycans for MS analysis is useful in assessing the overall glycan population, it requires more sample, time, and manipulations
than the other two methods. MS analysis of the intact protein can give information about the protein's molecular weight and
glycan masses, and in combination with tandem MS, can confirm the combination of glycan building blocks in the observed masses.
The bottom-up strategy has advantages when there is more than one glycosylation site, or more sequence information is needed,
including information about where the glycan is attached. These data can usually be generated in a single liquid chromatography–tandem
mass spectrometry (LC–MS/MS) experiment, but the data analysis is more complex and time-consuming.
All three strategies were applied to TrypZean. First, the accurate molecular weight was established using a high-resolution
electrospray ionization - time-of-flight (ESI–TOF) mass spectrometer on the intact protein. The deconvoluted spectrum of recombinant
bovine trypsin (see Figure 1a) shows a peak with a mass of 23,294 Da, which is consistent with the theoretical molecular weight
of native bovine trypsin. In addition, seven glycoforms, labelled with the numbers 1 to 7, were observed (2).
Figure 1: (a) Deconvoluted mass spectrometry (MS) spectrum of intact TrypZean. The delta masses above the nonglycosylated
base peak correspond to glycan species; (b) matrix-assisted laser desorption/ionization mass spectrum of released permethylated
glycans from TrypZean; (c) electrospray ionization–MS spectrum of triply charged glycopeptide Ser70-Lys89. Peaks 1–7 in all
spectra correspond to the same glycan compositions.
Previous attempts to release the glycans on TrypZean by N-glycosidase F and N-glycosidase A had not been successful (1). The investigators speculated that the protein was O-glycosylated, because the normal procedures to release N-linked glycans did not work. The standard O-glycosylation release method of [beta]-elimination also failed to work. The glycans were ultimately released using a more
aggressive [beta]-elimination procedure and subsequently permethylated to enhance detection during matrix-assisted laser desorption/ionization
mass (MALDI MS) analysis. Analysis of released and permethylated glycans provided additional evidence of the presence of the
seven glycoforms (see Figure 1b). The release conditions used were so harsh that it remained unclear whether the glycosylation
was N- or O-linked.
To identify the site of glycosylation, LC–MS/MS analysis of glycopeptides was carried out. First, a sodium dodecyl sulfate
polyacrylamide gel (SDS-PAGE) separation was performed, as shown in Figure 2. The gel showed the presence of two distinct
forms of the protein. The lower band is consistent with the migration of the 23 kDa native bovine trypsin, and the upper band
corresponds to the glycosylated form. These two bands were excised, and peptide mapping carried out for each using LC–MS/MS.
Data from each band correlated well with the sequence of native bovine trypsin, giving sequence coverages greater than 85%.
One 20-residue tryptic peptide was absent from the peptide map of the upper, glycosylated, protein band, implying that the
glycosylation was likely contained within the sequence of this tryptic peptide, 70SIVHPSYNSNTLNNDIMLIK89 (2).
Figure 2: (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel bands excised for electrospray ionization
liquid chromatography–tandem mass spectrometry analysis; (b) sequence coverage of glycosylated protein band (upper) and nonglycosylated
To confirm this hypothesis, the LC-MS/MS data from the tryptic digest of the glycosylated band was manually searched for the
presence of glycopeptides. The glycan moieties of the seven observed glycopeptides all seem to be attached to the same tryptic
peptide (see Figure 1c), and had similar masses to those found in the intact molecular weight and released glycan analysis,
as shown in Table II.
Table II: Summary of identified glycans found in TrypZean.