8 Ultrafast Ionization and Fragmentation: From Small Molecules
177
peptide backbone, and the dominant product ion reflects only this phosphoric acid
loss, rather than a series of product ions containing sequence information. Phosphate group loss and position scrambling have been identified as being problematic
in CID-MS n studies [61].
The branch of proteomics that focuses on post-translational modification (PTM)
analysis frequently deals with these more “problematic” samples, where comprehensive structural analysis requires cleaving strong bonds while leaving more labile
bonds intact. A number of alternative ion activation methods have been introduced
to achieve this required non-thermal fragmentation. Electron capture dissociation
(ECD) [62] and electron transfer dissociation (ETD) [63, 64] activate the precursor
ions through the formation of an unstable radical. The subsequent radical-directed
fragmentation pathways are complementary to CID, and leave weakly bound PTMs
intact. Photodissociation of trapped peptides in the ultraviolet [65–69] and vacuum
ultraviolet [68, 70–74] regimes also generates MS/MS spectra that are similar to
ETD and ECD spectra, and rely on photon absorption for ion activation rather than
electron transfer [74, 75].
Fs-LID is a viable alternative to these non-statistical ion activation methods. FsLID differs from other laser-induced activation methods in that the laser is in the
near IR region, far from the electronic excitation transitions of peptides. The ion
activation is achieved through tunnel ionization, as discussed earlier. Upon ionization of a protonated peptide, the oxidized species formed is a distonic cation
[M + H] + → [M + H] 2+• , which is susceptible to both proton- and radical-directed
fragmentation pathways. As a result, fs-LID MS/MS spectra are often more information rich than CID spectra. While conservative predictions may expect product
ion cleavages to occur at or near the original site of radical formation, reactive radicals have actually been demonstrated to migrate upon formation within a peptide
cation. This means that the radical is mobile and that its migration is coupled with
rearrangements within the molecule [76]. This can give rise to backbone cleavages
and side chain losses that propagate several residues away from the initial radical site [77, 78]. This mechanism for ion activation is applicable to positive-mode
MS/MS analysis of protonated peptides in any charge state and does not require a
chromophore. Fs-LID is compatible with any ion trap mass spectrometer, and the interfacing of the laser can be done without compromising CID capability. Currently,
the amplified laser is setup on a large optical table, but as ultrafast technology improves, the size and cost of these laser systems will decrease, making them more
appealing. Ultimately, a compact femtosecond fiber laser could be brought into an
existing mass spectrometry facility to make fs-LID an option for routine MS/MS
analyses. Novel approaches to fiber laser design, for example self-similar evolution
[79], has allowed for the development of compact fiber oscillators delivering peak
power levels of 250 kW and 42 fs pulse duration [80].
Photofragmentation studies of biomolecules using UV radiation from nanosecond lasers led to the suggestion that the use of tunable fs-UV laser pulses might lead
to efficient and non-ergodic dissociation of large molecules [81]. However, limited
work has paired a femtosecond Ti:Sapphire laser with an ion trap mass spectrometer for such dissociation studies. Laarman et al. used a learning algorithm with pulse
177
peptide backbone, and the dominant product ion reflects only this phosphoric acid
loss, rather than a series of product ions containing sequence information. Phosphate group loss and position scrambling have been identified as being problematic
in CID-MS n studies [61].
The branch of proteomics that focuses on post-translational modification (PTM)
analysis frequently deals with these more “problematic” samples, where comprehensive structural analysis requires cleaving strong bonds while leaving more labile
bonds intact. A number of alternative ion activation methods have been introduced
to achieve this required non-thermal fragmentation. Electron capture dissociation
(ECD) [62] and electron transfer dissociation (ETD) [63, 64] activate the precursor
ions through the formation of an unstable radical. The subsequent radical-directed
fragmentation pathways are complementary to CID, and leave weakly bound PTMs
intact. Photodissociation of trapped peptides in the ultraviolet [65–69] and vacuum
ultraviolet [68, 70–74] regimes also generates MS/MS spectra that are similar to
ETD and ECD spectra, and rely on photon absorption for ion activation rather than
electron transfer [74, 75].
Fs-LID is a viable alternative to these non-statistical ion activation methods. FsLID differs from other laser-induced activation methods in that the laser is in the
near IR region, far from the electronic excitation transitions of peptides. The ion
activation is achieved through tunnel ionization, as discussed earlier. Upon ionization of a protonated peptide, the oxidized species formed is a distonic cation
[M + H] + → [M + H] 2+• , which is susceptible to both proton- and radical-directed
fragmentation pathways. As a result, fs-LID MS/MS spectra are often more information rich than CID spectra. While conservative predictions may expect product
ion cleavages to occur at or near the original site of radical formation, reactive radicals have actually been demonstrated to migrate upon formation within a peptide
cation. This means that the radical is mobile and that its migration is coupled with
rearrangements within the molecule [76]. This can give rise to backbone cleavages
and side chain losses that propagate several residues away from the initial radical site [77, 78]. This mechanism for ion activation is applicable to positive-mode
MS/MS analysis of protonated peptides in any charge state and does not require a
chromophore. Fs-LID is compatible with any ion trap mass spectrometer, and the interfacing of the laser can be done without compromising CID capability. Currently,
the amplified laser is setup on a large optical table, but as ultrafast technology improves, the size and cost of these laser systems will decrease, making them more
appealing. Ultimately, a compact femtosecond fiber laser could be brought into an
existing mass spectrometry facility to make fs-LID an option for routine MS/MS
analyses. Novel approaches to fiber laser design, for example self-similar evolution
[79], has allowed for the development of compact fiber oscillators delivering peak
power levels of 250 kW and 42 fs pulse duration [80].
Photofragmentation studies of biomolecules using UV radiation from nanosecond lasers led to the suggestion that the use of tunable fs-UV laser pulses might lead
to efficient and non-ergodic dissociation of large molecules [81]. However, limited
work has paired a femtosecond Ti:Sapphire laser with an ion trap mass spectrometer for such dissociation studies. Laarman et al. used a learning algorithm with pulse