TEMPO-Assisted Free Radical-Initiated Peptide Sequencing Mass Spectrometry (FRIPS MS) in Q-TOF and Orbitrap Mass Spectrometers: Single-Step Peptide Backbone Dissociations in Positive Ion Mode

present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference ). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research.
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title: Dissociation behavior of a bifunctional tempo-active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry.
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title: Gas-Phase Unimolecular Dissociation Reveals Dominant Base Property of Protonated Homocysteine Sulfinyl Radical Ions.
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title: 213 nm Ultraviolet Photodissociation on Peptide Anions: Radical-Directed Fragmentation Patterns
abstract: AbstractCharacterization of acidic peptides and proteins is greatly hindered due to lack of suitable analytical techniques. Here we present the implementation of 213 nm ultraviolet photodissociation (UVPD) in high-resolution quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. Radical-driven backbone fragmentation provides 22 distinctive fragment ion types, achieving the complete sequence coverage for all reported peptides. Hydrogen-deficient radical anion not only promotes the cleavage of Cα–C bond but also stimulates the breaking of N–Cα and C–N bonds. Radical-directed loss of small molecules and specific side chain of amino acids are detected in these experiments. Radical containing side chain of amino acids (Tyr, Ser, Thr, and Asp) may possibly support the N–Cα backbone fragmentation. Proline comprising peptides exhibit the unusual fragment ions similar to reported earlier. Interestingly, basic amino acids such as Arg and Lys also stimulated the formation of abundant b and y ions of the related peptide anions. Loss of hydrogen atom from the charge-reduced radical anion and fragment ions are rationalized by time-dependent density functional theory (TDDFT) calculation, locating the potential energy surface (PES) of ππ* and repulsive πσ* excited states of a model amide system.
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title: Structure of the observable histidine radical cation in the gas phase: a captodative alpha-radical ion.
abstract: Protein-based radicals play crucial roles in some of the greatest biosynthetic challenges in nature, including photosynthesis and substrate oxidation. Radical centers have been located on aromatic and sulfur-containing amino acid residues, as well as glycine residues. Invariably these charged or neutral radical species are generated through involvement of an adjacent metal cofactor. The positions of charge and spin in the radical cations are paramount for reactivity modulation and proton-coupled electron transfer, but obtaining structural details is difficult even for the simplest models. 2] Experiments in vacuo permit the investigation of intrinsic properties of radical cations in the absence of a reactivity-modulating environment. Radical cations of amino acids and peptides have been produced in vacuo by one-electron transfer in collision-induced dissociations (CIDs) of a ternary complex system comprising copper(II), an auxiliary ligand, and the amino acid or peptide. Such ternary complexes are efficiently generated by electrospray ionization, and probed downstream by using mass spectrometry (MS). Under appropriate conditions, CID of the complex yields the radical cation of the amino acid or peptide that can be isolated and trapped for spectroscopic interrogation. Herein, we report the first infrared multiple photon dissociation (IRMPD) spectroscopic experiments on a prototypical amino acid radical cation, HisC, and its ternary complex ion. In a recent article, Ke et al. showed that, by judicious choice of the auxiliary ligand, HisC of different stabilities are formed through CID of the ternary complex ion. In particular, the use of 2,2’:6’,2’’-terpyridine (tpy) as the ligand leads primarily to a HisC that is stable on the MS timescale and can be isolated and fragmented at a subsequent MS stage; by contrast, employing acetone as the ligand results in a metastable HisC and only its fragment ions are observed. Furthermore, the former, relatively stable HisC fragments by losing a water molecule to give [b1-H]C + and then CO to give [a1-H]C , whereas the latter, metastable HisC dissociates spontaneously by losing first CO2 to give the 4-ethaniminoimidazole radical cation, which then loses methanimine to give the 4-methyleneimidazole radical cation. Density functional theory (DFT) calculations at the (unrestricted) UB3LYP/6-311 + + G(d,p) level of theory predicted five low-energy HisC structures. Scheme 1 shows these structures with additional, new information on the barriers against their interconversions (see Figures S2 and S3 in the Supporting Information for details). Ke et al. postulated that the stable and metastable HisC are His5 (the structure at the global minimum) and His2, respectively. His5 is a captodative aradical ion that differs from the canonical His1 structure in having the a-CH hydrogen migrated to the imino nitrogen of the imidazole ring; His2 is best described as a 4-ethaniminoimidazole radical cation solvated by CO2. His2–His5 are all unconventional structures, and experimental verification of the HisC structure is highly desirable for confirmation of the key roles played by spin and charge delocalization in HisC stabilization.  1 compares the experimental IRMPD spectrum collected for HisC with the DFT-predicted IR spectra of His1–His5. It is apparent that only one predicted IR spectrum, that of His5, resembles the measured IRMPD spectrum. In particular, His5 is the only isomer predicted to exhibit two bands, 1596 and 1653 cm , which are assigned as NH2 scissoring and C=O stretching, respectively, that match the 1606 and 1666 cm 1 bands in the IRMPD spectrum. The lack of a strong band at around 1780–1790 cm 1 in the IRMPD spectrum rules out the presence of a significant fraction of His3 and His4. Similarly, His1 can be ruled out by the presence of the doublet, 1606 and 1666 cm , and the absence of spectroscopic details in the region of 1077– 1320 cm . His2 can be eliminated by the absence of peaks at around 810–820 cm 1 and by the low endothermicity against loss of the solvating CO2 (5 kcalmol ). We interpret the excellent match between the experimental IRMPD spectrum and the predicted IR spectrum of His5 to indicate that His5 is the only abundant species present. This degree of selectivity is feasible as His5 is positioned at the bottom of a deep well on the potential-energy surface of HisC. The barriers against His5 converting into the other His isomers and dissociating into [b1-H]C + are high (Scheme 1), [*] Dr. J. Zhao, Dr. C.-K. Siu, Y. Ke, Dr. U. H. Verkerk, Prof. A. C. Hopkinson, Prof. K. W. M. Siu Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street Toronto, ON M3J 1P3 (Canada) E-mail: kwmsiu@yorku.ca

title: Formation of Peptide Radical Ions through Dissociative Electron Transfer in Ternary Metal—Ligand—Peptide Complexes
abstract: The formation and fragmentation of odd-electron ions of peptides and proteins is of interest to applications in biological mass spectrometry. Gas-phase redox chemistry occurring during collision-induced dissociation of ternary metal–ligand–peptide complexes enables the formation of a variety of peptide radicals, including the canonical radical cations, M+*, radical dications, [M+H]2+*, radical anions, [M-2H]−* and phosphorylated radical cations. In addition, odd-electron peptide ions with well-defined initial location of the radical site are produced through side-chain losses from the radical ions. Subsequent fragmentation of these species provides information regarding the role of charge and location of the radical site on the competition between radical-induced and proton-driven fragmentation of odd-electron peptide ions. This account summarizes current understanding of the factors that control the efficiency of the intramolecular electron transfer (ET) in ternary metal–ligand–peptide complexes resulting in formation of odd-electron peptide ions. Specifically, we discuss the effect of the metal center, the ligand and the peptide structure on the competition between the ET, proton transfer (PT) and loss of neutral peptide and neutral peptide fragments from the complex. Fundamental studies of the structures, stabilities and the energetics and dynamics of fragmentation of these complexes are also important for detailed molecular-level understanding of photosynthesis and respiration in biological systems.

title: Comparing the gas-phase fragmentation reactions of protonated and radical cations of the tripeptides GXR
abstract: Electrospray ionization (ESI) mass spectrometry of methanolic solutions of mixtures of the copper salt (2,2′:6′,2″-terpyridine)copper(II) nitrate monohydrate ([Cu(II)(tpy)(NO 3 ) 2 ]·H 2 O) and a tripeptide GXR (where X = 1 of the 20 naturally occurring amino acids) yielded [Cu(II)(tpy)(GXR)] 2+ ions, which were then subjected to collision induced dissociation (CID). In all but one case (GRR), these [Cu(II)(tpy)(GXR)] 2+ ions fragment to form odd electron GXR + radical cations with sufficient abundance to examine their gas-phase fragmentation reactions. The GXR + radical cations undergo a diverse range of fragmentation reactions which depend on the nature of the side chain of X. Many of these reactions can be rationalized as arising from the intermediacy of isomeric distonic ions in which the charge (i.e. proton) is sequestered by the highly basic arginine side chain and the radical site is located at various positions on the tripeptide including the peptide back bone and side chains. The radical sites in these distonic ions often direct the fragmentation reactions via the expulsion of small radicals (to yield even electron ions) or small neutrals (to form radical cations). Both classes of reaction can yield useful structural information, allowing for example, distinction between leucine and isoleucine residues. The gas-phase fragmentation reactions of the GXR + radical cations are also compared to their even electron [GXR+H] + and [GXR+2H] 2+ counterparts. The [GXR+H] + ions give fewer sequence ions and more small molecule losses while the [GXR+2H] 2+ ions yield more sequence information, consistent with the ‘mobile proton model’ described in previous studies. In general, all three classes of ions give complementary structural information, but the GXR + radical cations exhibit a more diverse loss of small species (radicals and neutrals). Finally, links between these gas-phase results and key radical species derived from amino acids, peptides and proteins described in the literature are made.

title: Non-zwitterionic structures of aliphatic-only peptides mediated the formation and dissociation of gas phase radical cations.
abstract: We have investigated how the non-zwitterionic and zwitterionic structures of aliphatic-only tripeptides affect the formation and dissociation of peptide radical cations in the gas phase. The non-zwitterionic forms of the aliphatic-only peptides in their metal complexes play an important role in determining whether the electron transfer pathway predominates. We extended this study by synthesizing permanent non-zwitterionic and zwitterionic forms of aliphatic-only peptide radical cations and exploring their reactivities in the gas phase. Collision-induced dissociation spectra demonstrated the feasibility of generating both non-zwitterionic and zwitterionic forms. Radical cations in zwitterionic forms may indeed mediate the beta and gamma carbon-carbon bond cleavages of leucine and isoleucine side chains from the GlyGlyXle radical peptides; this feature allows leucine and isoleucine residues to be distinguished unambiguously.

title: Side chain chemistry mediates backbone fragmentation in hydrogen deficient peptide radicals.
abstract: A crown ether based, photolabile radical precursor which forms noncovalent complexes with peptides has been prepared. The peptide/precursor complexes can be electrosprayed, isolated in an ion trap, and then subjected to laser photolysis and collision induced dissociation to generate hydrogen deficient peptide radicals. It is demonstrated that these peptide radicals behave very differently from the hydrogen rich peptide radicals generated by electron capture methods. In fact, it is shown that side chain chemistry dictates both the occurrence and relative abundance of backbone fragments that are observed. Fragmentation at aromatic residues occurs preferentially over most other amino acids. The origin of this selectivity relates to the mechanism by which backbone dissociation is initiated. The first step is abstraction of a beta-hydrogen from the side chain, followed by beta-elimination to yield primarily a-type fragment ions. Calculations reveal that those side chains which can easily lose a beta-hydrogen correlate well with experimentally favored sites for backbone fragmentation. In addition, radical mediated side chain losses from the parent peptide are frequently observed. Eleven amino acids exhibit unique mass losses from side chains which positively identify that particular amino acid as part of the parent peptide. Therefore, side chain losses allow one to unambiguously narrow the possible sequences for a parent peptide, which when combined with predictable backbone fragmentation should lead to greatly increased confidence in peptide identification.

title: Tracking radical migration in large hydrogen deficient peptides with covalent labels: Facile movement does not equal indiscriminate fragmentation
abstract: Photodissociation of iodo-tyrosine modified peptides yields localized radicals on the tyrosine side chain, which can be further dissociated by collisional activation. We have performed extensive experiments on model peptides, RGYALG, RGYG, and their derivatives, to elucidate the mechanisms underlying backbone fragmentation at tyrosine. Neither acetylation nor deuteration of the tyrosyl phenolic hydrogen significantly affects backbone fragmentation. However, deuterium migration from the tyrosyl β carbon is concomitant with cleavage at tyrosine. Substitution of tyrosine with 4-hydroxyphenylglycine, which does not have β hydrogens, results in almost complete elimination of backbone fragmentation at tyrosine. These results suggest that a radical situated on the β carbon is required for a-type fragmentation in hydrogen-deficient radical peptides. Replacement of the αH of the residue adjacent to tyrosine with methyl groups results in significant diminution of backbone fragmentation. The initial radical abstracts an αH from the adjacent amino acid, which is poised to “rebound” and abstract the βH of tyrosine through a six-membered transition-state. Subsequent β-scission leads to the observed a-type backbone fragment. These results from deuterated peptides clearly reveal that radical migration in peptides can occur and that multiple migrations are not infrequent. Counterintuitively, close examination of all experimental results reveals that the probability for fragmentation at a particular residue is well correlated with thermodynamic radical stability. A-type fragmentation therefore appears to be most likely when favorable thermodynamics are combined with the relevant kinetic control. These results are consistent with ab initio calculations, which demonstrate that barriers to migration are significantly smaller in magnitude than probable dissociation thresholds.

title: Is dissociation of peptide radical cations an ergodic process?
abstract: This study presents a first detailed investigation of the energetics and dynamics of dissociation of peptide radical cations using a model system, in which the initial position of the radical site is well-defined. We demonstrate that fragmentation is dominated by bond cleavages that are remote from the initial position of the radical site and that all the dissociation channels are adequately described by the RRKM theory. Our findings suggest that fragmentation of peptide radical cations does not circumvent the ergodic assumption.

title: Electrospray tandem mass spectrometry analysis of S- and N-nitrosopeptides: Facile loss of NO and radical-induced fragmentation
abstract: The covalent addition of nitric oxide (NO) to protein thiols, a posttranslational modification termed S-nitrosation, is a ubiquitous event that modulates diverse cellular processes. The in vivo addition of NO to protein amines (N-nitrosation) has also been described and may similarly modify protein structure and function. While mass spectrometry has been employed for identification of nitrosoproteins, little is known about how S- and N-nitrosopeptides fragment. Such knowledge is important for its potential to inform on sites of protein nitrosation. Here we used electrospray tandem mass spectrometry to elucidate collision-induced dissociation (CID) features of S- and N-nitrosopeptide ions. We show that S- and N-nitrosopeptide ions readily lose NO, giving rise to species that contain thiyl and aminyl radicals, respectively. Fragmentation (MS3) of these radical peptide ions revealed an atypical pattern, characterized by the cleavage of select αC-C and N-αC bonds, rather than the more usual cleavage of amide bonds that result in b- and y-ions. These unanticipated fragmentation patterns are reconciled by radical-mediated abstraction of hydrogen from β-carbon followed by β-fragmentation. For thiyl radical peptides, we also observed dominant loss of SH and CH2SH from the Cys side-chain. Our findings provide new insights into the gas-phase chemistry of NO-modified peptide ions and suggest an unusual fragmentation pattern that may aid in future MS-based attempts to define the nitrosoproteome.

title: Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry.
abstract: The gas-phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o-TEMPO-Bz-conjugated peptides with an intra- and intermolecular disulfide bond was investigated using MS(n) tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF-α (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16-31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o-TEMPO-Bz-conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S-S or C-S bond was readily cleaved. The observed peptide backbone fragments included a-, c-, x- or z-types, which indicates that the radical-driven peptide fragmentation mechanism plays an important role in TEMPO-FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S-S or C-S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of •SH or •SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S-S bond, the abstraction of a hydrogen atom at C(β) by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at C(α) is suggested to lead to C-S bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH.

title: Formation and Dissociation of Phosphorylated Peptide Radical Cations
abstract: In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal–ligand phosphorylated peptide complexes [CuII(terpy)pM]·2+ and [CoIII(salen)pM]·+ [pM: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N,N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations (pM·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M – H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the pM·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H3PO4 loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H3PO4 from a prototypical model—N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

title: Residue-specific radical-directed dissociation of whole proteins in the gas phase.
abstract: The rapid identification of proteins from biological samples is critical for extracting useful information in proteomics studies. Mass spectrometry is one among the various methods of choice for achieving this task; however, current approaches are limited by a lack of chemical control over proteins in the gas phase. Herein, it is shown that modification of tyrosine to iodo-tyrosine followed by UV photodissociation of the carbon-iodine bond can be used to generate a radial site specifically at the modified residue. The subsequent dissociation of the protein is largely dominated by radical-directed reactions, including dominant backbone fragmentation at the modified tyrosine. If iodination of the protein is carried out under natively folded conditions, the modification and ultimate fragmentation can typically be isolated to a single tyrosine residue. Some secondary backbone cleavage in the immediate vicinity of the modified tyrosine also occurs, especially if proline is present. In the absence of a reactive tyrosine residue, similar chemistry occurs via iodination at histidine. Possible mechanisms which would lead to the observed a-type fragments at tyrosine and the secondary fragments at proline are discussed. A method for using this type of site-specific information to reduce database searching times in proteomics experiments by several orders of magnitude is outlined.

title: Elucidating the tertiary structure of protein ions in vacuo with site specific photoinitiated radical reactions.
abstract: A new method for identifying residue specific through space contacts as a function of protein secondary and tertiary structure in the gas phase is presented. Photodissociation of a non-native carbon-iodine bond incorporated into Tyr59 of ubiquitin yields a radical site specifically at that residue. The subsequent radical migration is shown to be highly dependent on the structure of the protein. Radical-directed dissociation (RDD) of low charge states, which adopt compact structures, generates backbone fragmentation that is prominently distributed throughout the protein sequence, including residues that are distant in sequence from Tyr59. Higher charge states of ubiquitin, which adopt elongated, unfolded structures, yield RDD that is primarily nearby in sequence to Tyr59. Regardless of which structure is probed, information at the residue-level is obtained by examining specific radical-donor and radical-acceptor pairs. The relative importance of a particular interaction pair for a specific conformation can be revealed by tracking the charge state dependence of the dissociation. Structurally important contact pairs exhibit strong and concerted changes in relative intensities as a function of charge state and can also be used to reveal structural features which persist among different protein structures. Moreover, incorporation of distance constraint information into molecular mechanics conformational searches can be used to drive the search toward relevant conformational space. Implementation of this approach has revealed highly stable, previously undiscovered structures for the +4 and +6 charge states of ubiquitin, which bear little resemblance to the crystal structure.

title: Charge-Directed Peptide Backbone Dissociations of o -TEMPO-Bz-C(O)-Peptides
abstract: In the present study, we report that the charge-directed (assisted) peptide dissociation products, such as b- and y-type pep- tide backbone fragments, were the major products in MS/MS and MS 3 applications of some o-TEMPO-Bz-C(O)-peptide ions, while radical-driven dissociation products, such as a/x and c/z-type fragments, were previously shown to be the major products in the free radical initiated peptide sequencing mass spectrometry (FRIPS MS). Those o-TEMPO-Bz-C(O)-peptides share a common feature in their sequences, that is, the peptides do not include an arginine residue that has the highest proton affinity among free amino acids. The appearance of b- and y-type fragments as major products in FRIPS MS can be understood in terms of the so-called "mobile-pro- ton model". When the proton is highly mobilized by the absence of arginine, the chare-directed peptide dissociation pathways appear to be more competitive than the radical-driven dissociation pathways, in our FRIPS experiments.

title: Photodissociation of TEMPO-modified peptides: new approaches to radical-directed dissociation of biomolecules.
abstract: Radical-directed dissociation of gas phase ions is emerging as a powerful and complementary alternative to traditional tandem mass spectrometric techniques for biomolecular structural analysis. Previous studies have identified that coupling of 2-[(2,2,6,6-tetramethylpiperidin-1-oxyl)methyl]benzoic acid (TEMPO-Bz) to the N-terminus of a peptide introduces a labile oxygen-carbon bond that can be selectively activated upon collisional activation to produce a radical ion. Here we demonstrate that structurally-defined peptide radical ions can also be generated upon UV laser photodissociation of the same TEMPO-Bz derivatives in a linear ion-trap mass spectrometer. When subjected to further mass spectrometric analyses, the radical ions formed by a single laser pulse undergo identical dissociations as those formed by collisional activation of the same precursor ion, and can thus be used to derive molecular structure. Mapping the initial radical formation process as a function of photon energy by photodissociation action spectroscopy reveals that photoproduct formation is selective but occurs only in modest yield across the wavelength range (300-220 nm), with the photoproduct yield maximised between 235 and 225 nm. Based on the analysis of a set of model compounds, structural modifications to the TEMPO-Bz derivative are suggested to optimise radical photoproduct yield. Future development of such probes offers the advantage of increased sensitivity and selectivity for radical-directed dissociation.

title: Side Chain Cleavage in TEMPO‐assisted Free Radical Initiated Peptide Sequencing (FRIPS): Amino Acid Composition Information#
abstract: In tandem mass spectrometry, side-chain losses are known to provide information on the presence of certain amino acid residues, which can improve peptide identification. In this study, we examine whether side-chain losses also occur in free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS). All four peptides examined here, i.e., ALPMHIR, YRPPGFSPFR, RPPGFSPYR, and YGGFLRRIRPKLK, showed extensive small group losses in the so-called (M• – X) region. Furthermore, peaks due to small group loss accompanying backbone fragmentations were also extensively observed. The observed small group losses, either radical or neutral side-chain losses, confirmed the presence of certain amino acids, even exhibiting signature peaks for isoleucine and leucine. In addition, mechanisms for small group losses in FRIPS MS are proposed.

title: Radical-driven peptide backbone dissociation tandem mass spectrometry.
abstract: In recent years, a number of novel tandem mass spectrometry approaches utilizing radical-driven peptide gas-phase fragmentation chemistry have been developed. These approaches show a peptide fragmentation pattern quite different from that of collision-induced dissociation (CID). The peptide fragmentation features of these approaches share some in common with electron capture dissociation (ECD) or electron transfer dissociation (ETD) without the use of sophisticated equipment such as a Fourier-transform mass spectrometer. For example, Siu and coworkers showed that CID of transition metal (ligand)-peptide ternary complexes led to the formation of peptide radical ions through dissociative electron transfer (Chu et al., 2000. ). The subsequent collisional activation of the generated radical ions resulted in a number of characteristic product ions, including a, c, x, z-type fragments and notable side-chain losses. Another example is the free radical initiated peptide sequencing (FRIPS) approach, in which Porter et al. and Beauchamp et al. independently introduced a free radical initiator to the primary amine group of the lysine side chain or N-terminus of peptides (Masterson et al., 2004. ; Hodyss et al., 2005 ). Photodetachment of gaseous multiply charged peptide anions (Joly et al., 2008. ) and UV photodissociation of photolabile radical precursors including a C-I bond (Ly & Julian, 2008. ; Ly & Julian, 2009. 1158) also provide another route to generate radical ions. In this review, we provide a brief summary of recent results obtained through the radical-driven peptide backbone dissociation tandem mass spectrometry approach.

title: Bromine isotopic signature facilitates de novo sequencing of peptides in free-radical-initiated peptide sequencing (FRIPS) mass spectrometry.
abstract: We recently showed that free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) assisted by the remarkable thermochemical stability of (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) is another attractive radical-driven peptide fragmentation MS tool. Facile homolytic cleavage of the bond between the benzylic carbon and the oxygen of the TEMPO moiety in o-TEMPO-Bz-C(O)-peptide and the high reactivity of the benzylic radical species generated in •Bz-C(O)-peptide are key elements leading to extensive radical-driven peptide backbone fragmentation. In the present study, we demonstrate that the incorporation of bromine into the benzene ring, i.e. o-TEMPO-Bz(Br)-C(O)-peptide, allows unambiguous distinction of the N-terminal peptide fragments from the C-terminal fragments through the unique bromine doublet isotopic signature. Furthermore, bromine substitution does not alter the overall radical-driven peptide backbone dissociation pathways of o-TEMPO-Bz-C(O)-peptide. From a practical perspective, the presence of the bromine isotopic signature in the N-terminal peptide fragments in TEMPO-assisted FRIPS MS represents a useful and cost-effective opportunity for de novo peptide sequencing.

title: Guanidination of lysine residue improves the sensitivity and facilitates the interpretation of free radical initiated peptide sequencing (FRIPS) mass spectrometry results
abstract: Lysine-containing peptides, which are often found in tryptic peptide mixtures, frequently undergo double conjugations with o -TEMPO-Bz-C(O)- groups in TEMPO-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry applications because of the facile conjugation reaction at the ɛ-primary amino group of the lysine side chain. Doubly conjugated peptides required an additional collisional activation step because they include two free radical initiators, which is not favorable for the applications of the FRIPS approach in practical peptide MS analysis. To avoid this issue, this study introduces a new strategy of guanidinating lysine-containing peptides prior to the o -TEMPO-Bz-C(O)- conjugation. The guanidination step was found to be readily incorporated into our TEMPO-assisted FRIPS procedure with high yield. More importantly, the guanidinated peptides were demonstrated to undergo radical-driven peptide backbone fragmentations, similar to peptides that lack a lysine residue. In summary, combined with the guanidination step, the TEMPO-assisted FRIPS MS offers another practical tool that can be used to analyze and characterize peptides.

title: Collision-induced dissociation (CID) of peptides and proteins.
abstract: The most commonly used activation method in the tandem mass spectrometry (MS) of peptides and proteins is energetic collisions with a neutral target gas. The overall process of collisional activation followed by fragmentation of the ion is commonly referred to as collision-induced dissociation (CID). The structural information that results from CID of a peptide or protein ion is highly dependent on the conditions used to effect CID. These include, for example, the relative translational energy of the ion and target, the nature of the target, the number of collisions that is likely to take place, and the observation window of the apparatus. This chapter summarizes the key experimental parameters in the CID of peptide and protein ions, as well as the conditions that tend to prevail in the most commonly employed tandem mass spectrometers.

title: Higher-energy C-trap dissociation for peptide modification analysis
abstract: Peptide sequencing is the basis of mass spectrometry–driven proteomics. Here we show that in the linear ion trap–orbitrap mass spectrometer (LTQ Orbitrap) peptide ions can be efficiently fragmented by high-accuracy and full-mass-range tandem mass spectrometry (MS/MS) via higher-energy C-trap dissociation (HCD). Immonium ions generated via HCD pinpoint modifications such as phosphotyrosine with very high confidence. Additionally we show that an added octopole collision cell facilitates de novo sequencing.

title: Orbitrap mass spectrometry: instrumentation, ion motion and applications.
abstract: Since its introduction, the orbitrap has proven to be a robust mass analyzer that can routinely deliver high resolving power and mass accuracy. Unlike conventional ion traps such as the Paul and Penning traps, the orbitrap uses only electrostatic fields to confine and to analyze injected ion populations. In addition, its relatively low cost, simple design and high space-charge capacity make it suitable for tackling complex scientific problems in which high performance is required. This review begins with a brief account of the set of inventions that led to the orbitrap, followed by a qualitative description of ion capture, ion motion in the trap and modes of detection. Various orbitrap instruments, including the commercially available linear ion trap-orbitrap hybrid mass spectrometers, are also discussed with emphasis on the different methods used to inject ions into the trap. Figures of merit such as resolving power, mass accuracy, dynamic range and sensitivity of each type of instrument are compared. In addition, experimental techniques that allow mass-selective manipulation of the motion of confined ions and their potential application in tandem mass spectrometry in the orbitrap are described. Finally, some specific applications are reviewed to illustrate the performance and versatility of the orbitrap mass spectrometers.

title: Proposed nomenclature for peptide ion fragmentation
abstract: The multitude of fragmentation techniques available to modern tandem mass spectrometry introduces diversity in the types of product ions. An all-explicit nomenclature system for the product ions of peptide fragmentation is herein proposed for the sake of clarity and unambiguity. All variables – the charge, radical and hydrogens gained or lost – associated with a given product ion are specified.

title: Influence of Peptide Composition, Gas-Phase Basicity, and Chemical Modification on Fragmentation Efficiency: Evidence for the Mobile Proton Model
abstract: Relative energetics of fragmentation of protonated peptides are investigated by using electrospray ionization/surface-induced dissociation (ESI/SID) tandem mass spectrometry. ESI/SID fragmentation efficiency curves (percent fragmentation versus laboratory collision energy) are presented for 20 oligopeptides and are a measure of how easily a peptide fragments. The relative positions of the ESI/SID fragmentation efficiency curves depend on several parameters which include peptide composition (e.g., presence/absence of a basic amino acid residue) and peptide size. The ESI/SID fragmentation efficiency curves, in combination with quantum chemical calculations, provide a unique approach to substantiate and refine the mobile proton model for peptide fragmentation. Selected peptides are also investigated to further test and confirm the mobile proton model; these include doubly-protonated peptides and chemically-modified peptides (i.e., acetylated and fixed-charge derivatized peptides). Doubly-protonated peptides ...

title: Effect of the N-terminal basic residue on facile Cα-C bond cleavages of aromatic-containing peptide radical cations.
abstract: Fragmentation of radical cationic peptides [R(G)(n-2)X(G)(7-n)]˙(+) and [R(G)(m-2)XG]˙(+) (X = Phe or Tyr; m = 2-5; n = 2-7) leads selectively to a(n)(+) product ions through in situ C(α)-C peptide backbone cleavage at the aromatic amino acid residues. In contrast, substituting the arginine residue with a less-basic lysine residue, forming [K(G)(n-2)X(G)(7-n)]˙(+) (X = Phe or Tyr; n = 2-7) analogs, generates abundant b-y product ions; no site-selective C(α)-C peptide bond cleavage was observed. Studying the prototypical radical cationic tripeptides [RFG]˙(+) and [KFG]˙(+) using low-energy collision-induced dissociation and density functional theory, we have examined the influence of the basicity of the N-terminal amino acid residue on the competition between the isomerization and dissociation channels, particularly the selective C(α)-C bond cleavage viaβ-hydrogen atom migration. The dissociation barriers for the formation of a(2)(+) ions from [RFG]˙(+) and [KFG]˙(+)via their β-radical isomers are comparable (33.1 and 35.0 kcal mol(-1), respectively); the dissociation barrier for the charge-induced formation of the [b(2)- H]˙(+) radical cation from [RFG]˙(+)via its α-radical isomer (39.8 kcal mol(-1)) was considerably higher than that from [KFG]˙(+) (27.2 kcal mol(-1)). Thus, the basic arginine residue sequesters the mobile proton to promote the charge-remote selective C(α)-C bond cleavage by energetically hindering the competing charge-induced pathways.

title: Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS
abstract: AbstractWe have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (−)-ESI-MS, CID-MS/MS, and MS3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO–C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids.
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