Characterization of TrypZean: a Plant-Based Alternative to Bovine-Derived Trypsin (Peer-Reviewed) - An in-depth characterization of maize-derived trypsin revealed an unusual nonconsensus N-linked glyc

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Characterization of TrypZean: a Plant-Based Alternative to Bovine-Derived Trypsin (Peer-Reviewed)
An in-depth characterization of maize-derived trypsin revealed an unusual nonconsensus N-linked glycosylation.


BioPharm International
Volume 24, Issue 10, pp. 44-48

GLYCOSYLATION-SITE ANALYSIS


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.
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 (3).

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.


Figure 4: Pepsin cleavage chart of TrypZean glycopeptide Ser70–Lys89; the distribution of modifications, including glycosylation and oxidation, on various peptic peptides is listed.
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 5: Product-ion (MS3) spectrum of TrypZean glycopeptide Ser70–Asn77 (peptide + Hex3HexNAc2Xyl1Fuc1).
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.

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

PEER REVIEWED

Article submitted: Jul. 06, 2011.
Article accepted: Aug. 25, 2011.

REFERENCES

1. S.L. Woodard et al., Biotechnol. Appl. Biochem. 38, 123–130 (2003).

2. P.R. Jalili et al., Proceedings of the 56th ASMS Conference on Mass Spectrometry and Allied Topics (Denver, CO, 2008) pp. 1–5.

3. H. Zhang et al., Analytical Chem. 82 (24), 10095–10101 (2010).

4. E.E. Hood et al., NABC Report 17: Agricultural Biotechnology: Beyond Food and Energy to Health and the Environment (National Agricultural Biotechnology Council, Ithaca, NY, 2005) pp. 147–158.


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