Comparative studies of 193-nm photodissociation and TOF-TOFMS analysis of bradykinin analogues: The effects of charge site(s) and fragmentation timescales

Abstract

The dissociation reactions of [M ϩ H] ϩ , [M ϩ Na] ϩ , and [M ϩ Cu] ϩ ions of bradykinin (amino acid sequence RPPGFSPFR) and three bradykinin analogues (RPPGF, RPPGFSPF, PPGF-SPFR) are examined by using 193-nm photodissociation and post-source decay (PSD) TOF-TOF-MS techniques. The photodissociation apparatus is equipped with a biased activation cell, which allows us to detect fragment ions that are formed by dissociation of short-lived (Ͻ1 s) photo-excited ions. In our previously reported photodissociation studies, the fragment ions were formed from ions dissociating with lifetimes that exceeded 10 s; thus these earlier photofragment ion spectra and post-source decay (PSD) spectra [composite of both metastable ion (MI) and collision-induced dissociation (CID)] were quite similar. On the other hand, short-lived photo-excited ions dissociate by simple bond cleavage reactions and other high-energy dissociation channels. We also show that product ion types and abundances vary with the location of the charge on the peptide ion. For example, H ϩ and Na ϩ cations can bind to multiple polar functional groups (basic amino acid side chains) of the peptide, whereas Cu ϩ ions preferentially bind to the guanidino group of the arginine side-chain and the N-terminal amine group. Furthermore, when Cu ϩ is the charge carrier, the abundances of non-sequence informative ions, especially loss of small neutral molecules (H 2 O and NH 3 ) is decreased for both photofragment ion and PSD spectra relative to that observed for [ [3] have revolutionized the analysis of biomolecules by mass spectrometry (MS) techniques. Identification of proteins by using "bottom-up" MS techniques, i.e., enzymatic digestion of proteins or protein mixtures followed by peptide mass fingerprinting and database searching [4,5], is now routine, and de novo sequencing [6] and determination of post-translational modifications using tandem mass spectrometry techniques is now commonly practiced in many laboratories. The major factor that complicates peptide structure determination is that the types of fragment ions observed in a tandem mass spectrum are related to the intrinsic properties of the gas-phase peptide ion [7], the activation method used, and the timeframe for probing unimolecular dissociation products [8,9]. For example, utilizing only the internal energy acquired during desorption/ionization and a relatively long fragmentation timescale [ϳ10 s, as in post-source decay (PSD) experiments] favors product ions formed via the lowest energy channels [10], i.e., b i -and y i -type fragments [11,12]. As we showed previously, the observed fragment ions and their relative abundances change significantly if the timescale of the experiment is altered [13].Collisional activation of gas-phase peptide ions with a neutral gas (eV to several keV collision energies) can be employed to increase the internal energy of the ions and, thereby, enhance the abundances of sequence informative fragment ions; however, the resulting spectra also contain large numbers of non-se...

Factors that impact the vacuum ultraviolet photofragmentation of peptide ions

“…Two significant differences between the tandem-TOF and ion trap instruments could influence the photodissociation spectra that they produce. One is the observation timescale, as has been previously suggested [19,20]. In our tandem-TOF instrument, the time between photoexcitation and subsequent ion extraction into the second mass analyzer is about 1 s, while in the linear ion trap, the equivalent time is greater than 10 ms. A second significant difference involves buffer gas.…”

“…Since the clearest evidence of a unique fragmentation mechanism in 157 nm photodissociation comes from peptides with Cterminal arginine, these peptides should be the focus of comparative studies. In the previously described 193 nm photodissociation studies, only one peptide with a C-terminal arginine residue was investigated, and only y-type fragments were observed [19].…”

“…Morgan and Russell have compared 193 nm photodissociation in a tandem-TOF to their prior work using stepped reflectron voltages, and have studied some peptides with C-terminal arginine. Photodissociation spectra from the tandem-TOF were found to contain fewer b and y fragments [19]. Kim and coworkers have used the same wavelength to fragment several arginine-terminated peptides in a MALDI curved-field reflectron apparatus [24 -26].…”

Time-resolved photodissociation of singly protonated peptides with an arginine at the N-terminus: A statistical interpretation

“…Two important assumptions in the statistical theory of mass spectra, RRKM-QET (quasiequilibrium theory) [31,32], are rapid internal conversion to the ground electronic state and rapid intramolecular vibrational redistribution (IVR) in the ground electronic state. Whether the dissociation of an electronically excited peptide ion would satisfy the above two conditions has been a subject of controversy [21,23,27,33,34]. This question must be addressed before attempting to explain the appearance of three apparently different time-evolution patterns.…”

“…Since the main purposes of the previous work were to develop the new technique and to get a spectral overview, peptide ions with an arginine residue-there is some controversy on their dissociation mechanism [21,23,27]-were intentionally excluded in the study. With the working principle of the technique well established, we attempted to obtain and analyze the timeresolved PD data for peptide ions with an arginine residue at the N-terminus.…”