For peptides, absorption above 220 nm occurs through specific chromophores
provided by the side chains of aromatic amino acids. In nature, only three amino
acids (tryptophan, tyrosine and phenylalanine) have an aromatic ring system and
are thus accessible to near-UV excitation [3]. Spectroscopy of peptides and proteins
has mainly focused on sequences containing one of these three amino acids using
different laser schemes (UV, IR–UV, UV–UV and even IR–IR–UV multipleresonance methods. For a review see [4]). In addition to the absorption by aromatic
amino acids, different classes of proteins are coloured due to the presence of
prosthetic groups containing visible chromophores. The groups of S. Brøndsted
Nielsen and L.H. Andersen have pioneered the spectroscopy and photophysics of
isolated natural biochromophores [5, 6]. While the absorbance of coloured proteins
is highly sensitive to the prosthetic environment and absorption spectroscopy is an
essential tool to monitor the conformational changes and the associated dynamics
of proteins in solution, spectra of chromophores embedded in gas-phase proteins
have only been reported recently [7].
This chapter focuses on UV spectroscopy of peptide and protein ions. After a
short discussion of experimental setups and mechanisms, it explores the influence
of biomolecule conformation, chromophore environment, chromophore electronic
structure, and solvation on optical spectra.
8.2
Implementation of UV–Vis Action Spectroscopy
Ion-trap mass spectrometers, in which ions are stored in a specific place for a period
of time, are particularly well-suited for action-spectroscopy experiments. Trapped
ions are irradiated by a laser beam introduced to the instrument through a window
or access holes (see Fig. 8.1).
Results shown in this chapter were obtained using nanosecond-pulsed tuneable
optical parametric oscillators, with frequency doubling provided by barium borate
(BBO) crystals which allows us to scan the photon wavelength from 215 nm to
2 μm.
In order to record action spectra, mass spectra were recorded as a function of
laser wavelength. The yield of fragmentation at each laser wavelength is given by ln
((parent + ∑ frag)/parent)/ϕ, where ϕ is the laser fluence, parent is the intensity of
the parent peak and Σfrag represents the total intensity of the photofragment peaks.
Quadrupole ion traps allow the use of hybrid activation that combines photoexcitation, possibly with different laser beams, and collisional excitation. Different
schemes can be designed that combine the different excitations in the same or
different mass spectrometry stages. In addition to the instruments developed in our
laboratory [8, 9], different light sources can be used, including dye lasers and
synchrotron radiation [10]. Different ion-storage devices were also used for action
spectroscopy. Early experiments were performed with an FT-ICR instrument [11]
and an electrostatic storage ring [5]. A direction being taken at present is the
implementation of multipole ion traps for spectroscopy of cooled ions [4, 12].
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R. Antoine and P. Dugourd
provided by the side chains of aromatic amino acids. In nature, only three amino
acids (tryptophan, tyrosine and phenylalanine) have an aromatic ring system and
are thus accessible to near-UV excitation [3]. Spectroscopy of peptides and proteins
has mainly focused on sequences containing one of these three amino acids using
different laser schemes (UV, IR–UV, UV–UV and even IR–IR–UV multipleresonance methods. For a review see [4]). In addition to the absorption by aromatic
amino acids, different classes of proteins are coloured due to the presence of
prosthetic groups containing visible chromophores. The groups of S. Brøndsted
Nielsen and L.H. Andersen have pioneered the spectroscopy and photophysics of
isolated natural biochromophores [5, 6]. While the absorbance of coloured proteins
is highly sensitive to the prosthetic environment and absorption spectroscopy is an
essential tool to monitor the conformational changes and the associated dynamics
of proteins in solution, spectra of chromophores embedded in gas-phase proteins
have only been reported recently [7].
This chapter focuses on UV spectroscopy of peptide and protein ions. After a
short discussion of experimental setups and mechanisms, it explores the influence
of biomolecule conformation, chromophore environment, chromophore electronic
structure, and solvation on optical spectra.
8.2
Implementation of UV–Vis Action Spectroscopy
Ion-trap mass spectrometers, in which ions are stored in a specific place for a period
of time, are particularly well-suited for action-spectroscopy experiments. Trapped
ions are irradiated by a laser beam introduced to the instrument through a window
or access holes (see Fig. 8.1).
Results shown in this chapter were obtained using nanosecond-pulsed tuneable
optical parametric oscillators, with frequency doubling provided by barium borate
(BBO) crystals which allows us to scan the photon wavelength from 215 nm to
2 μm.
In order to record action spectra, mass spectra were recorded as a function of
laser wavelength. The yield of fragmentation at each laser wavelength is given by ln
((parent + ∑ frag)/parent)/ϕ, where ϕ is the laser fluence, parent is the intensity of
the parent peak and Σfrag represents the total intensity of the photofragment peaks.
Quadrupole ion traps allow the use of hybrid activation that combines photoexcitation, possibly with different laser beams, and collisional excitation. Different
schemes can be designed that combine the different excitations in the same or
different mass spectrometry stages. In addition to the instruments developed in our
laboratory [8, 9], different light sources can be used, including dye lasers and
synchrotron radiation [10]. Different ion-storage devices were also used for action
spectroscopy. Early experiments were performed with an FT-ICR instrument [11]
and an electrostatic storage ring [5]. A direction being taken at present is the
implementation of multipole ion traps for spectroscopy of cooled ions [4, 12].
142
R. Antoine and P. Dugourd