120
J. J. GORMAN et al.
attempt to obtain cytotoxic T lymphocytes capable of recognition of the peptide,
IMIKFNRL (residues 51-58), of the nuclear antigen La(SS-B), bound to major
histocompatability antigen molecules (Purcell et al. 1998). A peptide adduct
formed in relatively low yield as a byproduct during peptide synthesis appeared
to dominate the immunogenic response when the peptide preparation was used
as an immunogen.
3.1.1
Molecular Ion Analyses by MALDI-TOF-MS
The active agent involved in binding to major histocompatibility complex molecules such that the complex was recognised by a clonal line of cytotoxic T cells
was previously found to be a minor byproduct in the synthetic preparation of
residues 51-58 of the nuclear antigen La( SS-B) used for generation of the T cells
(Purcell et al. 1998). This byproduct and the target peptide (IMIKFNRL) were
isolated from the synthetic peptide mixture by reverse phase HPLC and their
masses determined by MALDI-TOF-MS (Fig. 8.1). The immunologically active
byproduct had a mono isotopic protonated molecular ion of m/z = 1090.61 (Fig.
8.lA) compared to the theoretical value of 1034.62 for the target sequence. The
isolated target peptide produced a protonated molecular ion of m/z = 1034.54
(Fig. 8.lB). This difference between the experimentally observed masses, 56 Da,
is consistent with modification by a butyl group as described previously (Purcell
et al. 1998). It was previously shown that the byproduct yielded an anomalous
PTH -amino acid derivative at position 6 during Edman sequencing indicating
that the Asn residue at position 6 was modified (Purcell et al. 1998). Thus, it was
postulated that the byproduct was modified due to formation of an N~-butyl
amide on Asn6 during the synthetic process, which would add 56Da as opposed
to a butyl ester which would add 57Da. Consequently, a peptide derivative was
deliberately synthesized with this modification and this derivative was found to
have the same mass (Fig. 8.1 C) as the immunologically active byproduct (Fig.
8.lA). Parenthetically, it should be noted that the deliberately synthesized derivative was found to have the same immunological characteristics as the isolated byproduct (Purcell et al. 1998).
The original synthetic product also contained a stable imide involving Asn6 of
the sequence which yielded a mixture of a- and ~-linked aspartyl pep tides upon
treatment with mild base. These aspartyl peptides were isolated and characterized by Edman degradation so as to differentiate the a- and ~-linked forms. The
a-aspartyl peptide produced a molecular ion IDa larger than the Asn6 form of
the peptide (Fig. 8.lD).
Fig. 8.2. PSD spectra of (A) the immunologically active synthetic byproduct, (B) the Asn6 version of
IMIKFNRL, (C) deliberately synthesised the Nj3-butyl-Asn6 version of IMIKFNRL and (D) the Asp6
version ofIMIKFNRL. Ions are labelled as belonging to C-terminal (yn and zn) or N-terminal (an, bn
and cn) series or as the immonium ion of Nj3-butyl-Asn (indicated by an asterisk) as defined in Fig.
8.3 and Table 8.1. Only ions derived by single peptide backbone bond cleavages are labelled, other
ions may have arisen as a consequence of multiple fragmentations and/or side chain cleavages. Positive symbols denote ions 56 Da heavier than fragments predicted for unmodified IMIKFNRL. These
spectra were collected using continuous extraction and a digitization rate of 250 MHz
J. J. GORMAN et al.
attempt to obtain cytotoxic T lymphocytes capable of recognition of the peptide,
IMIKFNRL (residues 51-58), of the nuclear antigen La(SS-B), bound to major
histocompatability antigen molecules (Purcell et al. 1998). A peptide adduct
formed in relatively low yield as a byproduct during peptide synthesis appeared
to dominate the immunogenic response when the peptide preparation was used
as an immunogen.
3.1.1
Molecular Ion Analyses by MALDI-TOF-MS
The active agent involved in binding to major histocompatibility complex molecules such that the complex was recognised by a clonal line of cytotoxic T cells
was previously found to be a minor byproduct in the synthetic preparation of
residues 51-58 of the nuclear antigen La( SS-B) used for generation of the T cells
(Purcell et al. 1998). This byproduct and the target peptide (IMIKFNRL) were
isolated from the synthetic peptide mixture by reverse phase HPLC and their
masses determined by MALDI-TOF-MS (Fig. 8.1). The immunologically active
byproduct had a mono isotopic protonated molecular ion of m/z = 1090.61 (Fig.
8.lA) compared to the theoretical value of 1034.62 for the target sequence. The
isolated target peptide produced a protonated molecular ion of m/z = 1034.54
(Fig. 8.lB). This difference between the experimentally observed masses, 56 Da,
is consistent with modification by a butyl group as described previously (Purcell
et al. 1998). It was previously shown that the byproduct yielded an anomalous
PTH -amino acid derivative at position 6 during Edman sequencing indicating
that the Asn residue at position 6 was modified (Purcell et al. 1998). Thus, it was
postulated that the byproduct was modified due to formation of an N~-butyl
amide on Asn6 during the synthetic process, which would add 56Da as opposed
to a butyl ester which would add 57Da. Consequently, a peptide derivative was
deliberately synthesized with this modification and this derivative was found to
have the same mass (Fig. 8.1 C) as the immunologically active byproduct (Fig.
8.lA). Parenthetically, it should be noted that the deliberately synthesized derivative was found to have the same immunological characteristics as the isolated byproduct (Purcell et al. 1998).
The original synthetic product also contained a stable imide involving Asn6 of
the sequence which yielded a mixture of a- and ~-linked aspartyl pep tides upon
treatment with mild base. These aspartyl peptides were isolated and characterized by Edman degradation so as to differentiate the a- and ~-linked forms. The
a-aspartyl peptide produced a molecular ion IDa larger than the Asn6 form of
the peptide (Fig. 8.lD).
Fig. 8.2. PSD spectra of (A) the immunologically active synthetic byproduct, (B) the Asn6 version of
IMIKFNRL, (C) deliberately synthesised the Nj3-butyl-Asn6 version of IMIKFNRL and (D) the Asp6
version ofIMIKFNRL. Ions are labelled as belonging to C-terminal (yn and zn) or N-terminal (an, bn
and cn) series or as the immonium ion of Nj3-butyl-Asn (indicated by an asterisk) as defined in Fig.
8.3 and Table 8.1. Only ions derived by single peptide backbone bond cleavages are labelled, other
ions may have arisen as a consequence of multiple fragmentations and/or side chain cleavages. Positive symbols denote ions 56 Da heavier than fragments predicted for unmodified IMIKFNRL. These
spectra were collected using continuous extraction and a digitization rate of 250 MHz