Elucidation of Functionally Significant Structural Modifications
127
FAB-MS was used for the original characterization of the process of cleavage
activation of the FO-proteins of common isolates of NDV and of some more
unusual isolates (Gorman et al. 1988, 1990a; Hodder et al. 1994; Scanlon et al.
1999). However, this required considerable quantities of starting material and
complementary use of Edman sequencing and amino acid analysis. In a recent
study MALDI-TOF-MS was used to characterize a variant isolate ofNDV that did
not react with the available antipeptide antibodies (Lopaticki et al. 1998). This
proved to be a much more comprehensive and sensitive approach than FAB-MS.
In particular, PSD was a valuable technique for characterizing the cleavage signalling motif of this isolate. In the MALDI-TOF-MS study under current consideration (Lopaticki et al. 1998) the F2-polypeptide of an immunologically unreactive avirulent isolate (VRI 82-6409) (Hodder et al. 1994) was isolated and compared to the F2-polypeptide of a well characterized avirulent strain (Queensland
or V4) (Simmons 1967) ofNDV.
F2-polypeptides were isolated from both strains of NDV by SDS-PAGE followed by electro elution (Gorman et al. 1988, 1990a). These F2-polypeptides were
digested with AspN protease and the unfractionated digests analysed by MALDITOF-MS using internal calibration (Fig. 804). A difference in mass between the Cterminal peptides of the two F2-polypeptides was apparent with the C-terminal
peptide of the VRI 82-6409 isolate (Fig. 8AA) being 72 Da heavier than that of
the V4 isolate (Fig. 8AB). Such mass difference is consistent with variation of one
of the glycine residues at the C-terminus of the V4 F2-polypeptide to glutamic
acid in the VRI isolate. These two AspN peptides were isolated by HPLC and analysed by PSD (Fig. 8.5; Table 8.2) which confirmed this difference and located the
variant residues as the C-terminal glycine of the V 4 isolate. Placement of the variation within the last three C-terminal residues was possible based on the fact that
a series of C-terminal ions was observed from y3 to y18 for the VRI 82-6409 isolate (Fig. 8.5A; Table 8.2) with masses 72 Da greater than masses predicted and/or
observed for the V4 isolate (Fig. 8.5B; Table 8.2). Definitive placement of the variation at the C-terminus was based on observation ofN-terminal ions for the VRI
82-6409 isolate (Fig. 8.5A; Table 8.2), up to and including the b18 fragment, with
masses the same as N-terminal fragments predicted and/or observed for the V4
isolate (Fig. 8.5B; Table 8.2).
3.3
Determination of the Disulphide Linkage Pattern in the Ectodomain
of the Respiratory Syncytial Virus Attachment Protein
Respiratory syncytial virus (RSV), which is another member of the paramyxoviridae family of viruses, causes serious lower respiratory infections of humans and
animals (Collins et al. 1996). Children up to 2 years of age are particularly susceptible to RSV and infection during this period is the expected norm (Collins et
al. 1996). In some instances hospitalization may be required as a consequence of
infection and mortality may occur in extreme cases. In order to combat RSV better it would be of benefit to understand the structural features of the RSV proteins involved in the early stages of cellular infection. Proteins of interest include
the viral attachment protein and the fusion protein. The attachment protein is a
127
FAB-MS was used for the original characterization of the process of cleavage
activation of the FO-proteins of common isolates of NDV and of some more
unusual isolates (Gorman et al. 1988, 1990a; Hodder et al. 1994; Scanlon et al.
1999). However, this required considerable quantities of starting material and
complementary use of Edman sequencing and amino acid analysis. In a recent
study MALDI-TOF-MS was used to characterize a variant isolate ofNDV that did
not react with the available antipeptide antibodies (Lopaticki et al. 1998). This
proved to be a much more comprehensive and sensitive approach than FAB-MS.
In particular, PSD was a valuable technique for characterizing the cleavage signalling motif of this isolate. In the MALDI-TOF-MS study under current consideration (Lopaticki et al. 1998) the F2-polypeptide of an immunologically unreactive avirulent isolate (VRI 82-6409) (Hodder et al. 1994) was isolated and compared to the F2-polypeptide of a well characterized avirulent strain (Queensland
or V4) (Simmons 1967) ofNDV.
F2-polypeptides were isolated from both strains of NDV by SDS-PAGE followed by electro elution (Gorman et al. 1988, 1990a). These F2-polypeptides were
digested with AspN protease and the unfractionated digests analysed by MALDITOF-MS using internal calibration (Fig. 804). A difference in mass between the Cterminal peptides of the two F2-polypeptides was apparent with the C-terminal
peptide of the VRI 82-6409 isolate (Fig. 8AA) being 72 Da heavier than that of
the V4 isolate (Fig. 8AB). Such mass difference is consistent with variation of one
of the glycine residues at the C-terminus of the V4 F2-polypeptide to glutamic
acid in the VRI isolate. These two AspN peptides were isolated by HPLC and analysed by PSD (Fig. 8.5; Table 8.2) which confirmed this difference and located the
variant residues as the C-terminal glycine of the V 4 isolate. Placement of the variation within the last three C-terminal residues was possible based on the fact that
a series of C-terminal ions was observed from y3 to y18 for the VRI 82-6409 isolate (Fig. 8.5A; Table 8.2) with masses 72 Da greater than masses predicted and/or
observed for the V4 isolate (Fig. 8.5B; Table 8.2). Definitive placement of the variation at the C-terminus was based on observation ofN-terminal ions for the VRI
82-6409 isolate (Fig. 8.5A; Table 8.2), up to and including the b18 fragment, with
masses the same as N-terminal fragments predicted and/or observed for the V4
isolate (Fig. 8.5B; Table 8.2).
3.3
Determination of the Disulphide Linkage Pattern in the Ectodomain
of the Respiratory Syncytial Virus Attachment Protein
Respiratory syncytial virus (RSV), which is another member of the paramyxoviridae family of viruses, causes serious lower respiratory infections of humans and
animals (Collins et al. 1996). Children up to 2 years of age are particularly susceptible to RSV and infection during this period is the expected norm (Collins et
al. 1996). In some instances hospitalization may be required as a consequence of
infection and mortality may occur in extreme cases. In order to combat RSV better it would be of benefit to understand the structural features of the RSV proteins involved in the early stages of cellular infection. Proteins of interest include
the viral attachment protein and the fusion protein. The attachment protein is a