Product ion yields in postsource decay and photodissociation at 193 and 266 nm were measured for some peptide ions without a basic amino acid residue ([Y(6) + H](+), [F(5) + H](+), and [YPFVEPI + H](+)) generated by matrix-assisted laser desorption ionization (MALDI). Data indicated statistical nature for the dissociation processes. Assuming that peptide ions formed by MALDI are in thermal equilibrium at temperature T and that their dissociation rate constants are specified by the critical energy (E(0)) and entropy (DeltaS(double dagger)), a method based on kinetic analysis was devised to determine these parameters simultaneously. The matrix used was found to affect the effective temperature of peptide ions, 2,5-dihydroxybenzoic acid (400-430 K) < sinapinic acid (440 K) < alpha-cyano-4-hydroxycinnamic acid (460-510 K), in agreement with previous perceptions. E(0) of around 0.6 eV and DeltaS(double dagger) of -24 eu were smaller than previous quantum chemical results for small model peptide ions.

Photodissociation at 193 nm of some singly protonated peptides generated by matrix-assisted laser desorption/ionization was investigated using tandem time-of-flight mass spectrometry. For peptides with arginine at the C-terminus, x, upsilon, and w fragment ions were generated preferentially while a and d fragment ions dominated for peptides with arginine at the N-terminus. These are the same characteristics as photodissociation at 157 nm reported previously. Overall, the photodissociation spectra obtained at 157 and 193 nm were strikingly similar.

In-source decay (ISD) and post-source decay (PSD) of a peptide ion ([Y 6 ϩ H] ϩ ) and a preformed ion (benzyltriphenylphosphonium, BTPP) generated by matrix-assisted laser desorption ionization (MALDI) were investigated with time-of-flight mass spectrometry. ␣-Cyano-4-hydroxycinammic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were used as matrices. For both ions, ISD yield was unaffected by delay time, indicating rapid termination of ISD. This was taken as evidence for rapid expansion cooling of hot "early" plume formed in MALDI. CHCA was hotter than DHB for [Y 6 ϩ H] ϩ while the matrix effect was insignificant for BTPP. The "early" plume temperature estimated utilizing previous kinetic results was 800 -900 K, versus 400 -500 K for "late" plume. The results support our previous finding that the temperature of peptide ions interrogated by tandem mass spectrometry was lower than most rough estimates of MALDI temperature. -5] is widely used to generate gas-phase ions from biomolecules, details of the processes involved are not firmly understood. Outstanding problems include the internal energy of ions formed by MALDI and how it is affected by matrix [2][3][4][5]. With the assumption of quasi-thermal equilibrium, the first problem becomes equivalent to the question on effective temperature [6 -11]. In post-source decay (PSD) [12] of peptide ions generated by MALDI with two common matrices, ␣-cyano-4-hydroxycinammic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), the relative product ion yield is in the order DHB Ͻ CHCA [7,13], suggesting the same order for the temperature. Accordingly, CHCA and DHB are often called "hot" and "cold" matrices, respectively. In our recent kinetic studies [14 -19] for peptide ions without arginine, which mostly generate a, b, and y type product ions, not only relative PSD yield but also photodissociation (PD) rate constant followed the order DHB Ͻ CHCA, in agreement with the above classification.Matrix effect on in-source decay (ISD) of peptide and small protein ions has also been investigated [11, 20 -

Photodissociation (PD) at 193 nm of various singly protonated peptides was investigated. These include peptides with an arginine residue at the C-terminus, N-terminus, at both termini, inside the chain, and those without an arginine residue. Monoisotopomeric selection was made for the precursor ions. Interference from the post-source decay (PSD) product signals was reduced as much as possible by using the deflection system (reported previously) and subtracting the remaining signals from the laser-on signals. The presence of an arginine residue and its position inside the peptide were found to significantly affect the PD spectra, as reported previously. Presence of a proline, aspartic acid, or glutamic acid residue hardly affected the PD spectral patterns. By comparing the PD spectra obtained at a few different wavelengths, it is concluded that the dissociation of the photoexcited ions occurs in their ground electronic states. Tentative explanations for the observed spectral correlations based on the statistical picture for the reactions are also presented. is a useful technique to study the structure and fragmentation mechanism of gas-phase ions. With the development of various techniques to generate gasphase ions from condensed-phase samples such as electrospray ionization (ESI) [2] and matrix-assisted laser desorption ionization (MALDI) [3], MS/MS has been widely used to study biological molecules, and most importantly, peptides and proteins.Ions formed with sufficient internal energy in the source may dissociate after exiting the source. This is called the metastable ion decomposition [4]. Post-source decay (PSD) [5] is the terminology for a similar phenomenon observed in tandem time-of-flight (TOF) mass spectrometry for ions that are generated by MALDI. Ions may be further activated to expedite their dissociation. Collisionally activated dissociation (CAD) [6,7], infrared multiphoton dissociation (IRMPD) [8], surfaceinduced dissociation (SID) [9,10], and electron capture dissociation (ECD) [11,12] are widely used for this purpose.PSD, low-energy CAD and SID, and IRMPD of peptide ions have been popular in the bottom-up strategy [13] for protein identification and de novo sequencing [14]. In these schemes, both ion activation and dissociation occur in the ground electronic state of the precursor ion. It is well known that the critical energy is one of the two most important parameters (the other is the entropy of activation) that affect the rate of a statistical reaction [15,16]. Dissociation of peptide ions using the above MS/MS schemes usually results in yand b-type product ions, which are the ions formed via reaction paths with small critical energy, and a-type ions with lesser intensity and frequency. It is also known that b-type ions are prominent with an arginine residue at the N-terminus of the peptide while y-type ions are prominent with the same residue at the Cterminus [17]. Some residue-specific dissociations have also been noticed, and they may be utilized for spectral interpretation. These include...

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-evolution of product ion signals in ultraviolet photodissociation (UV-PD) of singly protonated peptides with an arginine at the N-terminus was investigated by using a tandem time-of-flight mass spectrometer equipped with a cell floated at high voltage. Observation of different time-evolution patterns for different product ion types-an apparently nonstatistical behavior-could be explained within the statistical framework by invoking consecutive formation of some product ions and broad internal energy distributions for precursor ions. a n ϩ 1 and b n ions were taken as the primary product ions from this type of peptide ions. Spectral characteristics in post-source decay, UV-PD, and collisionally activated dissociation at low and high kinetic energies could be explained via rough statistical calculation of rate constants. Specifically, the striking characteristics in high-energy CAD and UV-PD-dominance of a n and d n formed via a n ϩ 1-were not due to the peculiarity of the excitation processes themselves, but due to quenching of the b n channels caused by the presence of arginine. ( T andem mass spectrometry has been widely used for identification and sequence determination of polypeptides and proteins [1][2][3]. In spite of extensive studies made so far, fundamental understanding on the dissociation of activated peptide ions observed by tandem mass spectrometry is still lacking. This is in contrast with the case of the dissociation of small polyatomic ions, for which nearly quantitative theoretical description and even prediction are possible [4]. The main reasons for such a lack of fundamental understanding are experimental and computational difficulties to get structural, mechanistic, and kinetic data for large polyatomic systems consisting of more than 100 atoms. In this regard, it is to be mentioned that we recently developed a systematic method to calculate sequence-specific statistical (Rice-Ramsperger-KasselMarcus, RRKM) rate constants for dissociations of peptide and protein ions [5][6][7].The most popular method to activate a peptide ion and hence to induce its dissociation is the collisionally activated dissociation (CAD) [8 -12]. The kinetic energy of a peptide ion is an important factor affecting the CAD spectral pattern. Hence, CAD of a peptide ion is classified into two categories, low (around 100 eV) and high (higher than 1 keV) energy regimes. In the lowenergy CAD [11], which is typically done with triple quadrupole ion trap and ion cyclotron resonance mass spectrometers, a peptide ion gains sufficient energy for dissociation usually via multiple collisions, each collision supplying rather small amounts of internal energy through vibrational excitation. Most of the product ions are b and y types (see Scheme 1) formed via rearrangement reactions.Presence of arginine, proline and aspartic acid residues and the charge state of a peptide ion are known to affect the relative intensities of these product ions. The "mobile proton" model [13,14] has been devised to explain this. In the high-energy CA...

With matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, total abundance of product ions formed by dissociation inside (in-source decay, ISD) and outside (post-source decay, PSD) the source was measured for peptide ions [Y 5 (R) with H for the ionizing proton). α-Cyano-4-hydroxycinammic acid was used as matrix. Product abundance became smaller in the presence of basic residues (H, K, and R), in the order Y 9 H ≈ K 9 R. In particular, product abundances in ISD of peptide ions with R were smaller than those with H or K by an order of magnitude, which, in turn, were smaller than that for [Y 6 + H] + by an order of magnitude. Product abundance was affected by the most basic residue when more than one basic residue was present. A kinetic explanation for the data was attempted under the assumption of quasi-thermal equilibrium for peptide ions in MALDI plume which undergoes expansion cooling. Dramatic disparity in product abundance was found to arise from small difference in critical energy and entropy. Results indicate similar transition structures regardless of basic residues present, where the ionizing proton keeps interacting with a basic site. Further implication of the results on the dissociation mechanism along b-y channels is discussed.

Product ion yields in post-source decay and time-resolved photodissociation at 193 and 266 nm were measured for some peptide ions with a histidine residue ([HF 6 ϩ H] ϩ , [F 6 H ϩ H] ϩ , and [F 3 HF 3 ϩ H] ϩ ) formed by matrix-assisted laser desorption ionization (MALDI). Compared with similar data for peptide ions without any basic residue reported previously, significant reduction in dissociation efficiency was observed. Internal temperatures (T) of the peptide ions and their dissociation kinetic parameters-the critical energy (E 0 ) and entropy (⌬S ‡ )-were determined by the method reported previously. Slight decreases in E 0 , ⌬S ‡ , and T were responsible for the histidine effect-reduction in dissociation rate constant. Regardless of the presence of the residue, ⌬S ‡ was far more negative than previous quantum chemical results. Based on this, we propose the existence of transition structures in which the nitrogen atoms in the histidine residue or at the N-terminus coordinate to the reaction centers. Reduction in T in the presence of a histidine residue could not be explained based on popular models for ion formation in MALDI, such as the gas-phase proton transfer model. T andem mass spectral information-list of product ions formed from a precursor ion and their relative intensities-is tremendously useful for peptide and protein sequencing [1]. Pioneering studies on the product ion species formed from protonated peptides generated by fast atom bombardment and the mechanistic explanations for their formation were made by Martin and Biemann [2]. Extensive investigations followed [3][4][5], which were made mostly for protonated peptides generated by matrix-assisted laser desorption ionization (MALDI) [6] and electrospray ionization (ESI) [7]. For small model peptide ions, investigations beyond simple mechanistic interpretation have been reported, such as the quantum chemical search for reaction paths [8 -10]. However, there has not been much study on kinetics of peptide ion dissociation [11].Tandem mass spectral patterns for protonated peptides are affected by factors, such as the charge state, the number of arginine residue, and the energy regime [2,3,12,13]. For singly protonated peptides-these will be called peptide ions from now on-without an arginine residue, b and y types (see reference [2] for product ion symbols) are the major product ions regardless of the energy regime. Oxazolone pathways [4, 8 -10] have been proposed to explain their formation, which consist of the migration of the additional proton to an amide nitrogen, rate-determining cleavage of the protonated amide bond via a five-membered ring transition structure, formation of a proton-bound dimer of an oxazolone derivative and a smaller peptide, and its breakup. For peptide ions with an arginine residue, b/y channels (rearrangement) are dominant in the lowenergy regime [3], such as in low-energy collisionally activated dissociation (CAD) and post-source decay (PSD). In the high-energy regime, such as in highenergy CAD [2] and ultraviolet p...