Ultrafast Ionization and Fragmentation: From Small Molecules to Proteomic Analysis

Proteomic analysis offers great diagnostic relevance, because unlike DNA, different cells in an organism express different proteins. In fact, the cellular proteome can vary as a function of time or in response to stimuli. Beyond amino acid sequence, protein function depends on chemical modifications known as posttranslational modifications (PTMs) that serve as “switches” and “signals” that activate or inhibit vital functions. Despite advances in mass spectrometry, which have led to the development of fully automated protein sequencing instruments, the mapping of PTMs remains a challenge. The interaction of intense near-infrared femtosecond laser pulses with isolated molecules or ions leads to the creation of radicalion species through an ultrafast process known as tunnel ionization. The resulting unstable ions fragment according to predictable dissociation pathways. Progress analyzing and controlling the fundamental processes taking place during photoionization and fragmentation of small polyatomic molecules has led to the development of femtosecond laser-induced ionization/dissociation (fs-LID) for proteomic analysis. Fs-LID has been proven effective for the mapping of phosphorylation sites as well as other PTMs along the peptide backbone. The fundamental steps involved in fsLID, which permits cleavage of strong bonds while leaving chemically labile bonds intact, are discussed. Numerous examples are given to illustrate this exciting new ion activation method, and potential applications are identified. 8.1 Ultrafast Field Ionization and Its Application to Analytical Chemistry The utility of ultrafast photoionization in analytical chemistry stems from the mechanism by which energy is deposited into the population of molecules or ions being studied. While IR laser excitation is comparable to a slow-heating method, and UV laser excitation relies on resonant photon absorption, the femtosecond laser can cause ultrafast electron loss (oxidation) through a process known as tunnel ionM. Dantus (B) · C.L. Kalcic Michigan State University, East Lansing, MI, USA e-mail: [email protected] R. de Nalda, L. Bañares (eds.), Ultrafast Phenomena in Molecular Sciences, Springer Series in Chemical Physics 107, DOI 10.1007/978-3-319-02051-8_8, © Springer International Publishing Switzerland 2014 171 172 M. Dantus and C.L. Kalcic Fig. 8.1 [5] Tunnel Ionization of Tyrosine. When an ultrafast laser pulse passes by the target molecule or ion in the gas phase, the intense electric field deforms the potential felt by electrons within the molecule. As a result, the electron that is most polarizable is able to escape, leaving behind a photoionized radical site ization [1]. Tunnel ionization is achieved when an electron, pulled by the electric field of the laser pulse, acquires sufficient energy to overcome its binding energy within a single optical cycle. This process is illustrated in Fig. 8.1. For an excitation wavelength near 800 nm, tunnel ionization requires a peak power density of 1014 W/cm2 and pulse duration shorter than 35 fs. These estimates are based upon reported ionization thresholds for small molecules in an intense laser field, and have been generalized for larger molecules [2–4]. Lasers, especially those with UV and VUV wavelengths, have been used to induce bond photodissociation. Unfortunately, the most accessible chromophores present in biomolecules have a wide range of absorption maxima, as illustrated in Fig. 8.2. Therefore, wavelength tuning is typically necessary to optimize the photofragmentation process of different analytes. Unlike conventional photodissociation, tunnel ionization relies only on the presence of a polarizable electron, not a specific chromophore. In this sense, under tunneling ionization conditions, the femtosecond laser can serve as a universal excitation source. Laser induced ionization has been a powerful method for studying the spectroscopy of weakly fluorescent molecules. When carried out with nanosecond laser pulses, ionization takes place through intermediate states that are resonant with the laser pulse energy. Given that most organic compounds have an ionization potential near 9 eV, ionization typically requires three UV photons. Such spectroscopic measurements are typically referred to as 2 + 1 resonantly enhanced multiphoton processes (REMPI). The use of short (< 100 fs) pulses with near-IR wavelengths opens a new path for ionization that is less dependent on resonance excitation of intermediate states. The transition from multiphoton ionization (MPI) to tunneling ionization 8 Ultrafast Ionization and Fragmentation: From Small Molecules 173 Fig. 8.2 The absorption maxima for several chromophores are plotted and grouped by classes of biomolecules. Note that larger pigments like chlorophyll have broad absorption spectra, and only the maxima are indicated in this figure in atoms was studied by Mevel et al. who observed that distinct MPI features in the photoelectron spectra (separated by the photon energy hv) gradually disappear as tunneling ionization becomes dominant [6]. Similar work on large polyatomic molecules (benzene, naphthalene and anthracene), revealed broad featureless photoelectron spectra stretching up to 25 eV [7]. The larger the molecule, the smoother the spectrum, indicating that tunneling ionization is the dominant mechanism for above threshold ionization. The conditions of that study were 1013 W/cm2, 780 nm, 170 fs. Based on those observations, the conditions of our experiments (larger molecules and much shorter pulses) place our approach in the tunneling ionization regime. Tunnel ionization is advantageous for analytical applications because it removes the need for wavelength tuning. Furthermore, as will be shown, tunnel ionization leads to ultrafast photodissociation processes that occur on a timescale faster than energy randomization. Therefore, tunnel ionization offers the ability to cleave strong bonds while leaving weaker bonds intact. 8.2 Mass Spectrometry Coupled to an Ultrafast Laser Source