Development of a time-resolved method for photodissociation mechanistic study of protonated peptides: Use of a voltage-floated cell in a tandem time-of-flight mass spectrometer

Abstract

Photodissociation at 266 nm of some protonated peptides was investigated using a tandem-TOF spectrometer equipped with a cell near its first time focal point where the laser was irradiated. When a high voltage was applied to the cell, each product ion peak split into several components with different flight times. One of these was due to in-cell direct formation of the product ion and another due to post-cell formation. Those in between were due to consecutive dissociations, the first steps of which occurred inside the cell and the second steps outside the cell. [3] has been established as a useful method to determine the structures of biomolecules. In this method, a precursor ion to be investigated is selected and induced to dissociate. Recording product ions generated from the selected precursor ion results in a tandem mass spectrum. Various ion activation techniques are used to induce ion dissociation such as, for example, collisionally activated dissociation (CAD) [4,5], surface-induced dissociation [6,7], infrared multiphoton dissociation [8], and electron capture-induced dissociation [9,10]. CAD is probably the most popular among these techniques. Dissociation of a precursor ion after exiting the ion source may occur without additional activation. This metastable ion decomposition is often called post-source decay (PSD) [11] when observed with tandem time-of-flight (TOF) mass spectrometers.Even though photoexcitation with visible or ultraviolet (UV) light has been practiced in the study of the structure and the dissociation dynamics of small molecular ions [12,13], its application to large biomolecules has been a relatively new development [14 -24]. Recently, we constructed two tandem TOF mass spectrometers and investigated photodissociation (PD) of singly protonated peptides generated by UV-MALDI [20,23,25]. Lasers with 266 and 193 nm were used for PD, which will be called UV-PD [20 -22]. In one instrument [20,25], a cylindrically focused laser beam was crossed with a high kinetic energy ion beam for photoexcitation near a time focal point of the instrument. By synchronizing the laser beam pulse with the timefocused ion beam pulse, monoisotopomeric selection could be achieved. In another instrument [23], such a delicate synchronization was given up in favor of the post-acceleration/delayed extraction method to achieve high resolution for product ions. Quality of the PD spectra obtained with these instruments was comparable to that of CAD spectra. Also, it was observed that PD spectra of singly protonated peptides were similar to the corresponding CAD spectra obtained in keV kinetic energy regime, or high-energy CAD spectra. That is, most of the spectral correlations in the dissociation of singly protonated peptides observed by Biemann and coworkers [26, 27] using a four-sector magnetic instrument were applicable to UV-PD.Understanding the mechanisms and kinetics in the dissociation of a gas-phase ion is one of the fundamental subjects in mass spectrometry. The standard method to elucidate ion dissociatio...

Time-resolved photodissociation study of singly protonated peptides with a histidine residue generated by matrix-assisted laser desorption ionization: Dissociation rate constant and internal temperature

“…Rather, large product ions formed by PD may have further dissociated to smaller product ions, as found in our previous time-resolved, viz. voltage-on, PD studies [17,21].…”

“…We also mentioned that not only the dissociations resulting in I but also the first-step dissociations for C occur inside the cell, while those resulting in P occur outside. P components can be easily identified [21], as marked in the expanded spectra shown in the figure. An instrument-based method to identify I and the intermediate ion (m i ϩ ) contributing to each C has been developed recently [26] [18] as demonstrated in Figure 6.…”

“…A systematic method to calculate k(E) for peptide ions is available [19,20]. Regardless, rigorous RRKM analysis of tandem mass spectral data for peptide ions is a formidable problem because many competing and consecutive reactions participate [21], requiring more than one set of (E 0 , ⌬S ‡ ) for the kinetic analysis. Assuming that the kinetic properties of b/y channels occurring at different amide bonds are similar, the total rate constant, k tot (E), for peptide ions without an arginine residue may be estimated by calculating k(E) for a typical (average) channel and multiplying the result by the number of amide bonds-the approach taken in our previous study [18].…”

Investigations of Some Liquid Matrixes for Analyte Quantification by MALDI

“…Two homebuilt MALDI-tandem TOF instruments [31,32] were used in this work. Each consists of an ion source with delayed extraction and a reflectron.…”

Observation of phosphorylation site-specific dissociation of singly protonated phosphopeptides

“…Other chemicals, including the matrix (DHB), were purchased from Sigma (St. Louis, MO, USA). The method to prepare MALDI samples was the same as described previously [24]. The method for H/D exchange was also reported previously [25].…”