The 20-amino acid tryptic peptide identified as the location of the glycosylation contained four asparagine residues, three
serines, and one threonine, any of which might have been glycosylated. Despite the best efforts using these standard characterization
techniques, the authors were unable to determine the precise nature of the glycosylation. Standard tandem MS spectra of glycopeptides
tend to yield fragments resulting from cleavage of the glycan rather than the peptide backbone, and this proved to be the
case here (see Figure 3). New electron-driven dissociation technology (ETD), which fragments the peptide backbone without
losing post-translational modifications such as glycosylation, was applied. The ETD data suggested that the glycosylation
was at Asn-77, however, this could not be definitively confirmed because of the incomplete fragment ion series that was obtained
Figure 3: Product-ion (CID) spectrum of TrypZean glycopeptide Ser70–Lys89 (peptide + Hex3HexNAc2Xyl1Fuc1). The triply charged
ion of m/z 1154.5253 was the precursor ion. Blue square is N-acetylglucosamine or HexNAc; red triangle is fucose or Fuc; green
circle is hexose or Hex; yellow star is xylose or Xyl.
In collaboration with Michael Gross's group at Washington University in St. Louis, MO, the authors developed a novel method
for preparing the sample that allowed the enzyme to be analyzed more precisely. The normal procedure for digesting a protein
for characterization involves using porcine trypsin to generate peptides. Instead, a novel sample preparation was investigated
that uses a different enzyme: pepsin. Unlike trypsin, which cuts at arginine and lysine residues only, pepsin has limited
specificity and produces smaller fragments that are more amenable to ionization and MS analysis.
The pepsin was used in immobilized form attached to agarose beads, and the tryptic glycopeptide was exposed to it for varying
amounts of time. The theory behind this strategy was that by exposing the glycopeptide briefly, a peptide with only the first
amino acid cleaved would be generated. Then, taking a sample a little later in the digestion, a peptide with the first two
residues gone could be generated, a longer digestion still would yield a peptide with the third residue cleaved, and so on.
By nibbling at the end of the peptide in this way and taking mass spectral data at each point, it would become clear when
the amino acid bearing the sugar was removed.
Figure 4: Pepsin cleavage chart of TrypZean glycopeptide Ser70–Lys89; the distribution of modifications, including glycosylation
and oxidation, on various peptic peptides is listed.
By working down the amino acid sequence of the glycopeptide in this way, a series of 12 peptic fragments was generated (see
Figure 4). MS3 analysis of the various peptide fragments showed definitively that the glycan was attached to Asn-77 (see Figure
5). This result occurred despite the fact that the sequence was asparagine–serine–asparagine, which, according to the accepted
consensus sequence rules, should have precluded N-glycosylation.
Figure 5: Product-ion (MS3) spectrum of TrypZean glycopeptide Ser70–Asn77 (peptide + Hex3HexNAc2Xyl1Fuc1).
The small fragments created using this technique make it easy to identify with confidence the exact site of glycosylation
in cases such as this one where several possibilities exist. In straightforward cases, standard porcine trypsin digestion
remains adequate, but additional pepsin digestion should prove useful where multiple post-translational modifications or modification
sites occur. The less specific nature of pepsin digestion leads to a ladder series of fragments that can help definitively
identify the site of modification.
The combination of techniques used in this investigation allowed the authors to show definitively the location of the glycosylation
in TrypZean. As far as the authors are aware, this work is the first definitive experimental proof that a nonconsensus N-glycosylation occurs in maize-derived bovine trypsin. Small amounts of glycosylation may occur at other sites, but it is
evident that glycosylation at the Asn-77 residue is by far the most abundant.
Kevin Ray, PhD,* is a manager of analytical R&D and Pegah R. Jalili, PhD, is a senior R&D scientist in analytical R&D, both at SAFC. *To whom correspondance should be addressed
Article submitted: Jul. 06, 2011.
Article accepted: Aug. 25, 2011.
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