200
M. Dantus and C.L. Kalcic
then contribute to the −56 2+ product ion abundance observed in Fig. 8.17. Pathway
(b) follows the radical-directed cleavage of the C α –C β bond in the methionine side
chain, resulting in the neutral loss of 74 Da, corresponding to the −74 2+ product ion
peak in Fig. 8.17. Finally, Pathway (c) illustrates one possible outcome of the radical
migrating away from the methionine side-chain. The ion can now undergo a radicaldirected backbone cleavage of the N–C α bond in the peptide, resulting in c and/or
z-type ions. Note that this mechanism is non-specific and could occur at various
points along the peptide, giving rise to the z 3 and z 7 ions observed in Fig. 8.17. The
mechanism is shown at the methionine residue here only for simplicity, in reality
the resulting z 6 ion is a minor product ion and is not labeled in Fig. 8.17.
8.5 Discussion and Future Outlook
The ease with which femtosecond lasers are able to cause tunnel ionization in large
isolated molecules and ions has led to the development of tools that have application
in analytical chemistry. Already, fs-LID has been shown to generate informationrich MS/MS spectra. Through the analysis of protonated amino acids, we have identified the aromatic amino acids and methionine as the most likely sites of radical formation upon tunnel ionization, and have determined that ionization energy is not the
sole predictor of sample amenability to fs-LID. As the fs-LID MS/MS spectra of the
peptides illustrate, high polarizability and low proton mobility can boost fs-LID dissociation efficiency up above 35 %, even in samples with no aromatic chromophore
to enhance ion activation. This makes fs-LID an attractive ion activation technique
because it requires no chromophore or sample derivatization prior to MS/MS analysis. While a VUV laser can be used to efficiently photodissociate peptides due to
the absorption maximum of peptide bonds around 190 nm [96], a femtosecond laser
can activate any class of molecules via tunneling ionization, independent of their
absorption spectra.
Another benefit of using femtosecond laser pulses is their ability to open several non-ergodic dissociation pathways, wherein stronger bonds are broken and
more labile bonds remain intact. This observation has lead to a number of fundamental studies, and is a major attraction as an analytical tool. In particular,
non-ergodic dissociation is valuable for the analysis of post-translational modification. There is an alternative method for inducing this type of gas phase radical
known as electron capture or electron transfer dissociation (ECD/ETD), which reduces multiply charged gas phase ions to form hydrogen-abundant radical cations
([M + 2H] 2+ → [M + 2H] +• ). By comparison, fs-LID intermediates are hydrogendeficient ([M + H] + → [M + H] 2+• ), and therefore the technique does not need
a multiply charged precursor to carry out ion activation in positive ion mode. This
makes fs-LID appropriate for pairing with laser desorption sources such as MALDI,
as the soft ionization method is known to generate singly protonated ions. Preliminary studies in negative ion mode using fs-LID have also shown promising results
and higher product ion yields, indicating that fs-LID may one day be appropriate for
high throughput proteomic and metabolomic studies.
M. Dantus and C.L. Kalcic
then contribute to the −56 2+ product ion abundance observed in Fig. 8.17. Pathway
(b) follows the radical-directed cleavage of the C α –C β bond in the methionine side
chain, resulting in the neutral loss of 74 Da, corresponding to the −74 2+ product ion
peak in Fig. 8.17. Finally, Pathway (c) illustrates one possible outcome of the radical
migrating away from the methionine side-chain. The ion can now undergo a radicaldirected backbone cleavage of the N–C α bond in the peptide, resulting in c and/or
z-type ions. Note that this mechanism is non-specific and could occur at various
points along the peptide, giving rise to the z 3 and z 7 ions observed in Fig. 8.17. The
mechanism is shown at the methionine residue here only for simplicity, in reality
the resulting z 6 ion is a minor product ion and is not labeled in Fig. 8.17.
8.5 Discussion and Future Outlook
The ease with which femtosecond lasers are able to cause tunnel ionization in large
isolated molecules and ions has led to the development of tools that have application
in analytical chemistry. Already, fs-LID has been shown to generate informationrich MS/MS spectra. Through the analysis of protonated amino acids, we have identified the aromatic amino acids and methionine as the most likely sites of radical formation upon tunnel ionization, and have determined that ionization energy is not the
sole predictor of sample amenability to fs-LID. As the fs-LID MS/MS spectra of the
peptides illustrate, high polarizability and low proton mobility can boost fs-LID dissociation efficiency up above 35 %, even in samples with no aromatic chromophore
to enhance ion activation. This makes fs-LID an attractive ion activation technique
because it requires no chromophore or sample derivatization prior to MS/MS analysis. While a VUV laser can be used to efficiently photodissociate peptides due to
the absorption maximum of peptide bonds around 190 nm [96], a femtosecond laser
can activate any class of molecules via tunneling ionization, independent of their
absorption spectra.
Another benefit of using femtosecond laser pulses is their ability to open several non-ergodic dissociation pathways, wherein stronger bonds are broken and
more labile bonds remain intact. This observation has lead to a number of fundamental studies, and is a major attraction as an analytical tool. In particular,
non-ergodic dissociation is valuable for the analysis of post-translational modification. There is an alternative method for inducing this type of gas phase radical
known as electron capture or electron transfer dissociation (ECD/ETD), which reduces multiply charged gas phase ions to form hydrogen-abundant radical cations
([M + 2H] 2+ → [M + 2H] +• ). By comparison, fs-LID intermediates are hydrogendeficient ([M + H] + → [M + H] 2+• ), and therefore the technique does not need
a multiply charged precursor to carry out ion activation in positive ion mode. This
makes fs-LID appropriate for pairing with laser desorption sources such as MALDI,
as the soft ionization method is known to generate singly protonated ions. Preliminary studies in negative ion mode using fs-LID have also shown promising results
and higher product ion yields, indicating that fs-LID may one day be appropriate for
high throughput proteomic and metabolomic studies.