Elucidation of Functionally Significant Structural Modifications
131
type II integral membrane protein with a heavily glycosylated ectodomain that
contains four conserved cysteine residues (Collins et al. 1996). This protein is
termed the G-protein as a consequence of heavy glycosylation (Gruber and
Levine 1985).
MALDI-TOF-MS was used to characterize the disulfide bond arrangement of
human RSV G-protein directly isolated from virus infected cells (Gorman et al.
1997). HPLC of a tryptic digest of the G-protein revealed a predominant peak
of absorbance which was shown by MALDI-TOF-MS to contain ions of m/z =
4108.1 and 4125.4 (Fig. 8.6). These ions were consistent with the sequence spanning residues 152-187 provided no glycans were attached to any amino acid
side chains and the four cysteines within the sequence were in disulfide linkages. The mass difference of approximately 17 Da between these ions can be
accounted for by the fact that the amino terminus of the peptide is glutamine
which is susceptible to cyclization, through loss of ammonia, under the acidic
conditions of separation. Further digestion of the isolated tryptic peptide with
pepsin resulted in two peptides containing two disulfides each (Fig. 8.7). The
difference between these two peptic peptides was due solely to an additional
peptide bond cleavage in one of the peptides. This peptide consisted of two
disulfide-linked peptide chains with one of the chains containing an intrachain
disulfide bond in addition to the interchain disulfide. By comparison the other
peptic peptide, which lacked the additional peptide bond cleavage, had two
intrachain disulfide bonds.
PSD analysis of the two chain peptic peptide was used to determine that the
disulfide bonds of the G-protein were in a 1 to 4 plus 2 to 3 arrangement (Gorman et al. 1997). This peptide underwent extensive post-source fragmentation
that reflected cleavages along the peptide backbone (Fig. 8.8A; Table 8.3).
Although some disulfide bond fission was evident (Figs. 8.8B and 8.8C), the interchain peptide bond survived during production of the majority of the PSD ions.
Fragments resulting from peptide backbone cleavages were evident that effectively reflected sequential losses of amino acids along the backbone of the larger
of the two chains of this peptide up to the first of the three half cystines of the
larger chain. Thereafter, fragmentation reflected loss of the smaller peptide chain
in addition to the amino acid sequence of the larger chain (Fig. 8.8A). Peptide
backbone cleavages were apparent for the N-terminal portion of the larger peptide chain in the form ofN- and C-terminal ion series but did not extend into the
intrachain disulfide loop of this chain. Internal fragments were also evident (Figs.
8.8B and 8.8C; Table 8.3) that were consistent with location of the disulfides indicated by the sequential fragmentation data. The interchain disulfide was preserved within these internal fragments. Some fragmentation of the interchain
disulfide also occurred via both symmetric and asymmetric fragmentation (Figs.
8.8B and 8.8C; Table 8.3).
By comparison (Fig. 8.9), the single chain peptic peptide, with two intra chain
disulfide loops, only produced sequence information for the N-terminal amino
acids prior to the half-cystine proposed to participate in the interchain disulfide
linkage of the two chain peptic peptide (Fig. 8.9B). A single chain peptide derived
by post-proline cleavage of the original tryptic peptide, which commenced with
the half-cystine involved in the interchain disulfide in the two chain peptic pep-
131
type II integral membrane protein with a heavily glycosylated ectodomain that
contains four conserved cysteine residues (Collins et al. 1996). This protein is
termed the G-protein as a consequence of heavy glycosylation (Gruber and
Levine 1985).
MALDI-TOF-MS was used to characterize the disulfide bond arrangement of
human RSV G-protein directly isolated from virus infected cells (Gorman et al.
1997). HPLC of a tryptic digest of the G-protein revealed a predominant peak
of absorbance which was shown by MALDI-TOF-MS to contain ions of m/z =
4108.1 and 4125.4 (Fig. 8.6). These ions were consistent with the sequence spanning residues 152-187 provided no glycans were attached to any amino acid
side chains and the four cysteines within the sequence were in disulfide linkages. The mass difference of approximately 17 Da between these ions can be
accounted for by the fact that the amino terminus of the peptide is glutamine
which is susceptible to cyclization, through loss of ammonia, under the acidic
conditions of separation. Further digestion of the isolated tryptic peptide with
pepsin resulted in two peptides containing two disulfides each (Fig. 8.7). The
difference between these two peptic peptides was due solely to an additional
peptide bond cleavage in one of the peptides. This peptide consisted of two
disulfide-linked peptide chains with one of the chains containing an intrachain
disulfide bond in addition to the interchain disulfide. By comparison the other
peptic peptide, which lacked the additional peptide bond cleavage, had two
intrachain disulfide bonds.
PSD analysis of the two chain peptic peptide was used to determine that the
disulfide bonds of the G-protein were in a 1 to 4 plus 2 to 3 arrangement (Gorman et al. 1997). This peptide underwent extensive post-source fragmentation
that reflected cleavages along the peptide backbone (Fig. 8.8A; Table 8.3).
Although some disulfide bond fission was evident (Figs. 8.8B and 8.8C), the interchain peptide bond survived during production of the majority of the PSD ions.
Fragments resulting from peptide backbone cleavages were evident that effectively reflected sequential losses of amino acids along the backbone of the larger
of the two chains of this peptide up to the first of the three half cystines of the
larger chain. Thereafter, fragmentation reflected loss of the smaller peptide chain
in addition to the amino acid sequence of the larger chain (Fig. 8.8A). Peptide
backbone cleavages were apparent for the N-terminal portion of the larger peptide chain in the form ofN- and C-terminal ion series but did not extend into the
intrachain disulfide loop of this chain. Internal fragments were also evident (Figs.
8.8B and 8.8C; Table 8.3) that were consistent with location of the disulfides indicated by the sequential fragmentation data. The interchain disulfide was preserved within these internal fragments. Some fragmentation of the interchain
disulfide also occurred via both symmetric and asymmetric fragmentation (Figs.
8.8B and 8.8C; Table 8.3).
By comparison (Fig. 8.9), the single chain peptic peptide, with two intra chain
disulfide loops, only produced sequence information for the N-terminal amino
acids prior to the half-cystine proposed to participate in the interchain disulfide
linkage of the two chain peptic peptide (Fig. 8.9B). A single chain peptide derived
by post-proline cleavage of the original tryptic peptide, which commenced with
the half-cystine involved in the interchain disulfide in the two chain peptic pep-