Infrared Multiphoton Dissociation IRMPD

Infrared multiphoton dissociation (IRMPD) is used in FT-ICRMS instruments to generate fragmentations similar to those obtained by CID. IRMPD takes place 

The IRMPD experiment has been developed into a quantitative tool for the estimation of activation barriers of fragmentation processes, the so-called FRAGMENT method [28]. The barriers correspond to binding energies for simple bond dissociation reactions, if the reverse reactions do not have significant barriers. Consequently, kinetic and thermodynamic data are accessible with this method despite the ill-definition of temperature. The experiment relies on the generation of a steady state of IR photon absorption and emission after a short induction period. 

In this case, thermal equilibrium is reached and internal energies (at least for large ions) follow Boltzmann distributions. Where this equilibrium lies and which actual temperature the ions thus have is not known, but when the laser fiux density is increased, the steady state is shifted towards vibrationally more highly excited ions. Or expressed in a simplified way the ion temperature depends on the laser flux density. 

Practically, the rate constants fe for the dissociation of interest are determined at different laser flux densities P (given in W i.e. different ion temperatures. 

Fragmentations are unimolecular reactions, and thus, they follow the Arrhenius law (Eq. (5.9)). Since the actual temperature is unknown, the Arrhenius plot of In fe over l/T needs to be modified in that In k is plotted against In P. Linear relationsships are obtained. The activation energy Ea can be determined from the slope of the line. Ea contains the enthalpic contributions to the barrier. The entropic contributions remain unknown, because they are included in the preexponential factor A of the Arrhenius equation. This factor can only be determined from the intersection of the line with the ordinate and since the actual temperature is not known, there is no way to determine the intersection and the pre-exponential factor. 

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The activation step can alternatively be performed without gas by means of infrared multiphoton dissociation (IRMPD) or electron capture dissociation (BCD) (Chap. 2.12.2). Both IRMPD and BCD, solely require storage of the ions during their excitation by photons or electrons, respectively. It is one of the most charming properties of FT-ICR-MS/MS that even the accurate mass of the fragment ions can be determined. [216,217]... 

FT-ICR instruments are also capable of performing MS" experiments. The most popular method of ion activation is sustained off-resonance irradiation (SORI), where ions are excited to a larger cyclotron radius using rf energy, undergo collisions with a neutral gas pulsed into the cell and dissociate. Other methods are available, including infrared multiphoton dissociation (IRMPD)65 and electron capture dissociation (ECD)66 which is of particular value in glyco-peptide analysis (Section VIA). 

Two other ion activation methods were developed to replace the gas molecules as targets by laser beams (photodissociation or infrared multiphoton dissociation IRMPD) or by electron beams (electron capture dissociation ECD). These two methods can be applied to ions that are trapped during their excitations by photons or electrons, respectively. Thus, they are most often used with ion trap or ICR analysers because the residence time and the interaction time are longer. 

The fragmentation of peptides can also be obtained by FTICR instruments. Besides the most commonly used activation method, namely CID, the activation can alternatively be performed without gas by infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD). These methods fragment peptide ions in the ICR cell by emitting a laser beam or electron beam, respectively. 

Other fragmentation techniques have been introduced [108]. Some of these, e.g., sustained off-resonance irradiation (SORl) and infrared multiphoton dissociation (IRMPD), provide similar fragmentation as in CID, i.e., preferential backbone cleavages at the peptide amide bond (b- and y-ions). Others like electron-capture dissociation (ECD) [109-110] induce different fragmentation reactions, i.e., the formation of c- and z -ions due to cleavage of N-C bonds. 

To this end, different ion fragmentation tools have been characterized with respect to phosphopeptide fragmentation, e.g., electron-capture dissociation (ECD) [31] and infrared multiphoton dissociation (IRMPD) [32]. An application of ECD in PTM analysis is the top-down protein characterization (Ch. 18.3.5) of carbonic anhydrase [33]. IRMPD is applied in the study on protein kinase C phosphorylation [30]. Both ECD and IRMPD were applied in a subsequent nano-ESI-FT-ICR-MS study on protein kinase A phosphorylation [34]. Combined ECD and IRMPD for multistage MS-MS in FT-ICR-MS was applied for phosphopeptide characterization [35]. ECD provides backbone cleavages (c- and z -ions) without H3PO4 loss, whereas in IRMPD the loss of H3PO4 is prominent and only a few backbone cleavages (b- andy-ions) are observed cf. Ch. 17.6.1). 

Another ion activation method that is well suited for identification and sequence analysis of phosphopep-tides in the positive and negative polarity modes is infrared multiphoton dissociation (IRMPD).105,112 In this technique, phosphopeptides are irradiated with 10.6 pm photons emitted from a C02 laser. The phosphate group behaves like a chromophore for these photons, allowing evenly distributed cleavages in the peptide chain and more sequence coverage than the CID technique.105... 

This novel custom ESI-qQq-FT-ICR instrument allows the performance of several types of MS/MS experiments including Q2 collisionally activated dissociation (Q2 CAD), electron capture dissociation (ECD), and infrared multiphoton dissociation (IRMPD). 

Fragmentation of peptide and protein ions in FT-ICR mass spectrometry may be induced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) [28], infrared multiphoton dissociation (IRMPD) [29,30], blackbody infrared radiative dissociation (BIRD) [31,32], surface-induced dissociation (SID) [33,34], and electron capture dissociation (ECD) [35,36]. These techniques are true MS/MS techniques in which the precursor ion is isolated prior to fragmentation. Additional techniques in which ions are not isolated but fragmented before they... 

Infrared Multiphoton Dissociation (IRMPD) of Peptides and Proteins... 

ESI-Infrared Multiphoton Dissociation IRMPD of ESI-generated ions can also provide sequence information. By combining the data from the nozzle-skimmer dissociation and IRMPD experiments, nearly complete sequence coverage for larger oligonucleotides can be obtained [48]. These two dissociation techniques yield complementary sequence ions. For example, IRMPD primarily produces w- and (a — B )-type sequence ions, whereas the nozzle-skimmer dissociation generates b-, c-, and [Pg.467]

For MALDI ions, photodissociation is the next most common method for generating fragments. Here, a chromophore is required to absorb the laser light, because of which infrared multiphoton dissociation (IRMPD) [91] using a CO2 laser is preferred because such lasers emit in a wavelength where bending and rotational modes of biomolecules absorb. This is particularly true because the CO2 emission wavelengths are fairly broad, typicaUy from 9.1-10.6 im. Once the ions... 

Activation of the vibrational energy of ions can also be induced by the absorption of IR radiations. A popular type of IR radiation source is far-IR laser. In fact, many molecules have a broad IR absorption band. The most widely used IR source is a continuous wave (c.w.) CO2 laser, with the wavelength of 10.6 pm. This wavelength corresponds to an energy of 0.3 eV per laser photon. Because decomposition of a chemical bond requires >1 eV, laser excitation has to extended over hundreds of milliseconds to allow ions to absorb multiple IR photons. This method is known as infrared multiphoton dissociation (IRMPD). Another type of similar technique is black-body infrared radiative dissociation... 

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