118
J. J. GORMAN et al.
long drift time to the reflector (Kaufmann et al. 1993, 1994; Spengler et al. 1991,
1992). Protocols have been developed to analyse these metastable ions and enable
their interpretation in terms of peptide sequence data (Rouse et al. 1995; Spengler
1997). However, analysis of PSD fragmentation is not widely accepted as the
method of choice for sequencing peptides and other biopolymers. This is apparently due to a combination of the relative complexity of the experimental protocols used to obtain data, the inherent complexity of the data and the lack of universality of fragmentation with all samples of interest. Despite these reputed limitations, we have found PSD of MALD! generated ions has enabled characterization of structural features of modified peptides. The efficacy of PSD for biopolymer characterization will be exemplified below by elucidation of the structures of
modified pep tides and an oligosaccharide derivative. These include: a byproduct
of peptide synthesis that produced a dominant immunological response; variations in the sites of cleavage activation of the fusion protein precursors of different isolates of Newcastle disease virus (NDV); the disulfide bonding pattern of
the attachment protein of human respiratory syncytial virus (RSV); and, a 1phenyl-3-methyl-5-pyrazolone (PMP) derivative of pentaglucose.
2
Experimental
All data presented herein were acquired using a Bruker Reflex mass spectrometer
using experimental protocols described in detail elsewhere (Gorman et al. 1996,
1997; Lopaticki et al. 1998) or as elaborated in the legends to specific figures. Procedures for isolation of peptides for analysis have also been described in detail in
previous publications (Gorman et al. 1987, 1988, 1990b; Lopaticki et al. 1998) as
has the methodology for preparation and isolation of phenylmethylpyrazolone
derivatized oligosaccharides (Pitt and Gorman 1997).
3
Results and Discussion
3.1
Characterization of an N~-Butyl Asparagine Modified Peptide
with Immunodominance over the Unmodified Sequence
Synthetic peptides are frequently used as tools to study biological structure and
function (Kent 1988). However, complications can arise in such studies due to
synthetic bypro ducts in peptide preparations that can behave as superagonists or
inhibitors. Such byproducts can dominate the responsiveness of the biological
system and give rise to false interpretations. This was recently exemplified in an
Fig. 8.1. Parent ion masses of (A) the immunologically active byproduct formed during the synthesis
of IMIKFNRL, (B) the Asn6 version of IMIKFNRL, (e) deliberately synthesised the N~-butyl-Asn6
version of IMIKFNRL and (D) the a-Asp6 version of IMIKFDRL. These spectra were all collected
using delayed extraction and a digitization rate of IGHz
J. J. GORMAN et al.
long drift time to the reflector (Kaufmann et al. 1993, 1994; Spengler et al. 1991,
1992). Protocols have been developed to analyse these metastable ions and enable
their interpretation in terms of peptide sequence data (Rouse et al. 1995; Spengler
1997). However, analysis of PSD fragmentation is not widely accepted as the
method of choice for sequencing peptides and other biopolymers. This is apparently due to a combination of the relative complexity of the experimental protocols used to obtain data, the inherent complexity of the data and the lack of universality of fragmentation with all samples of interest. Despite these reputed limitations, we have found PSD of MALD! generated ions has enabled characterization of structural features of modified peptides. The efficacy of PSD for biopolymer characterization will be exemplified below by elucidation of the structures of
modified pep tides and an oligosaccharide derivative. These include: a byproduct
of peptide synthesis that produced a dominant immunological response; variations in the sites of cleavage activation of the fusion protein precursors of different isolates of Newcastle disease virus (NDV); the disulfide bonding pattern of
the attachment protein of human respiratory syncytial virus (RSV); and, a 1phenyl-3-methyl-5-pyrazolone (PMP) derivative of pentaglucose.
2
Experimental
All data presented herein were acquired using a Bruker Reflex mass spectrometer
using experimental protocols described in detail elsewhere (Gorman et al. 1996,
1997; Lopaticki et al. 1998) or as elaborated in the legends to specific figures. Procedures for isolation of peptides for analysis have also been described in detail in
previous publications (Gorman et al. 1987, 1988, 1990b; Lopaticki et al. 1998) as
has the methodology for preparation and isolation of phenylmethylpyrazolone
derivatized oligosaccharides (Pitt and Gorman 1997).
3
Results and Discussion
3.1
Characterization of an N~-Butyl Asparagine Modified Peptide
with Immunodominance over the Unmodified Sequence
Synthetic peptides are frequently used as tools to study biological structure and
function (Kent 1988). However, complications can arise in such studies due to
synthetic bypro ducts in peptide preparations that can behave as superagonists or
inhibitors. Such byproducts can dominate the responsiveness of the biological
system and give rise to false interpretations. This was recently exemplified in an
Fig. 8.1. Parent ion masses of (A) the immunologically active byproduct formed during the synthesis
of IMIKFNRL, (B) the Asn6 version of IMIKFNRL, (e) deliberately synthesised the N~-butyl-Asn6
version of IMIKFNRL and (D) the a-Asp6 version of IMIKFDRL. These spectra were all collected
using delayed extraction and a digitization rate of IGHz