8 Ultrafast Ionization and Fragmentation: From Small Molecules
195
while leucine, isoleucine, and proline could be photoionized upon derivatization (see
Table 8.1). Methionine is the exception in this category—the S heteroatom in the
side chain significantly lowers the ionization energy and accordingly, the activated
sample gives rise to strong fs-LID product ions in all protonated and derivatized
forms.
The fs-LID MS/MS analysis of single amino acids allowed us to elucidate the
most likely origin of the [M + H] 2+• ion-radical pair. Methioinine and the aromatic
residues are the most amenable to photoionization. Extending to slightly larger
molecular systems, the fs-LID product ion signal seems to track with ionization
potential or polarizability. Utilizing the benzoyl group as a chromophore was one
successful method for generating fs-LID product ions from previously inactive samples. However, this sort of wet chemistry is unnecessary if the analyte is sufficiently
large. The N-acetyl methyl ester derivatives were studied to mimic the lengthening
of the backbone in a longer peptide or protein, and this modification also led to the
observation of fs-LID product ions from previously inactive amino acids. Our conclusion is that longer peptides will be largely amenable to interrogation by fs-LID
regardless of their sequence, without the need for any derivatization prior to MS/MS
analysis.
8.4.6 Protein Sequencing
In proteomic MS/MS, the greater the number of assignable product ions observed,
the more information-rich the spectrum can be considered. Peptide sequencing can
be done manually or with the aid of software, whereby pairs of peaks that differ by
the mass of a single amino acid are used to map out the identity and order of residues
in the precursor sequence. If a specific region of the peptide does not fragment
well, the exact sequence in that cannot be assigned. While some of the product
ions corresponding to backbone cleavages may be redundant (in that we observe
multiple cleavages between the same pair of residues), they increase our confidence
in the ultimate sequence assignment. Therefore, it is advantageous to find an ion
activation method that yields a greater variety of product ions, rather than simply a
high intensity of product ions.
The robustness of fs-LID as an ion activation method is confirmed by the fs-LID
spectrum of the peptide GAILAGAILA, which contains no aromatic or methionine residues (Fig. 8.15). The polarizability of the large molecule is sufficient for
photoionization and gives rise to sufficient product ions for nearly 100 % sequence
coverage. The most abundant product ions are the −56 2+• (side chain loss from Leu
or Ile) and a nearly complete series of b-ions, limited only by the low mass cutoff
(LMCO) associated with isolation of the precursor at 869.4 Da.
A single residue substitution at the C-terminal end of the peptide from alanine
to arginine leads to a ∼7 % increase in dissociation efficiency by fs-LID (see Table 8.2) and also gives rise to more abundant a-ions near the C-terminal end of the
195
while leucine, isoleucine, and proline could be photoionized upon derivatization (see
Table 8.1). Methionine is the exception in this category—the S heteroatom in the
side chain significantly lowers the ionization energy and accordingly, the activated
sample gives rise to strong fs-LID product ions in all protonated and derivatized
forms.
The fs-LID MS/MS analysis of single amino acids allowed us to elucidate the
most likely origin of the [M + H] 2+• ion-radical pair. Methioinine and the aromatic
residues are the most amenable to photoionization. Extending to slightly larger
molecular systems, the fs-LID product ion signal seems to track with ionization
potential or polarizability. Utilizing the benzoyl group as a chromophore was one
successful method for generating fs-LID product ions from previously inactive samples. However, this sort of wet chemistry is unnecessary if the analyte is sufficiently
large. The N-acetyl methyl ester derivatives were studied to mimic the lengthening
of the backbone in a longer peptide or protein, and this modification also led to the
observation of fs-LID product ions from previously inactive amino acids. Our conclusion is that longer peptides will be largely amenable to interrogation by fs-LID
regardless of their sequence, without the need for any derivatization prior to MS/MS
analysis.
8.4.6 Protein Sequencing
In proteomic MS/MS, the greater the number of assignable product ions observed,
the more information-rich the spectrum can be considered. Peptide sequencing can
be done manually or with the aid of software, whereby pairs of peaks that differ by
the mass of a single amino acid are used to map out the identity and order of residues
in the precursor sequence. If a specific region of the peptide does not fragment
well, the exact sequence in that cannot be assigned. While some of the product
ions corresponding to backbone cleavages may be redundant (in that we observe
multiple cleavages between the same pair of residues), they increase our confidence
in the ultimate sequence assignment. Therefore, it is advantageous to find an ion
activation method that yields a greater variety of product ions, rather than simply a
high intensity of product ions.
The robustness of fs-LID as an ion activation method is confirmed by the fs-LID
spectrum of the peptide GAILAGAILA, which contains no aromatic or methionine residues (Fig. 8.15). The polarizability of the large molecule is sufficient for
photoionization and gives rise to sufficient product ions for nearly 100 % sequence
coverage. The most abundant product ions are the −56 2+• (side chain loss from Leu
or Ile) and a nearly complete series of b-ions, limited only by the low mass cutoff
(LMCO) associated with isolation of the precursor at 869.4 Da.
A single residue substitution at the C-terminal end of the peptide from alanine
to arginine leads to a ∼7 % increase in dissociation efficiency by fs-LID (see Table 8.2) and also gives rise to more abundant a-ions near the C-terminal end of the