Combining UV photodissociation action spectroscopy with electron transfer dissociation for structure analysis of gas‐phase peptide cation‐radicals

We report the first example of using ultraviolet (UV) photodissociation action spectroscopy for the investigation of gas‐phase peptide cation‐radicals produced by electron transfer dissociation. z ‐Type fragment ions ^●Gly‐Gly‐Lys⁺, coordinated to 18‐crown‐6‐ether (CE), are generated, selected by mass and photodissociated in the 200–400 nm region. The UVPD action spectra indicate the presence of valence‐bond isomers differing in the position of the C_α radical defect, (α‐Gly)‐Gly‐Lys⁺(CE), Gly‐(α‐Gly)‐Lys⁺(CE) and Gly‐Gly‐(α‐Lys⁺)(CE). The isomers are readily distinguishable by UV absorption spectra obtained by time‐dependent density functional theory (TD‐DFT) calculations. In contrast, conformational isomers of these radical types are calculated to have similar UV spectra. UV photodissociation action spectroscopy represents a new tool for the investigation of transient intermediates of ion‐electron reactions. Specifically, z ‐type cation radicals are shown to undergo spontaneous hydrogen atom migrations upon electron transfer dissociation. Copyright © 2015 John Wiley & Sons, Ltd.     

Ground and Excited-Electronic-State Dissociations of Hydrogen-Rich and Hydrogen-Deficient Tyrosine Peptide Cation Radicals

We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H]^+● and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS³ dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%–94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in Cu^II(2,2′:6′,2″-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR]^+● that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing C_α-radical positions in peptide cation radical intermediates.

Open image in new window

Graphical Abstract ?

     

Comparison of MS/MS Methods for Characterization of DNA/Cisplatin Adducts

The development of activation/dissociation techniques such as ultraviolet photodissociation (UVPD), infrared multiphoton dissociation (IRMPD), and electron transfer dissociation (ETD) as alternatives to collision induced dissociation (CID) has extended the range of strategies for characterizing biologically relevant molecules. Here, we describe a comprehensive comparison of CID, IRMPD, UVPD, ETD, and hybrid processes termed ETcaD and ET-IRMPD (and analogous hybrid methods in the negative mode NETcaD and NET-IRMPD) for generating sequence-specific fragment ions and allowing adduction sites to be pinpointed for DNA/cisplatin adducts. Among the six MS/MS methods, the numerous products generated by the IRMPD and UVPD techniques resulted in the most specific and extensive backbone cleavages. We conclude that IRMPD and UVPD methods generally offer the best characteristics for pinpointing the cisplatin adduction sites in the fragment-rich spectra.      

Dipolar DC induced collisional activation of non‐dissociated electron‐transfer products

The application of electron transfer and dipolar direct current induced collisional activation (ET‐DDC) for enhanced sequence coverage of peptide/protein cations is described. A DDC potential is applied across one pair of opposing rods in the high‐pressure collision cell of a hybrid quadrupole/time‐of‐flight tandem mass spectrometer (QqTOF) to induce collisional activation, in conjunction with electron transfer reactions. As a broadband technique, DDC can be employed for the simultaneous collisional activation of all the first‐generation charge‐reduced precursor ions (eg, electron transfer no‐dissociation or ETnoD products) from electron transfer reactions over a relatively broad mass‐to‐charge range. A systematic study of ET‐DDC induced collision activation on peptide/protein cations revealed an increase in the variety (and abundances) of sequence informative fragment ions, mainly c‐ and z‐type fragment ions, relative to products derived directly via electron transfer dissociation (ETD). Compared with ETD, which has low dissociation efficiency for low‐charge‐state precursor ions, ET‐DDC also showed marked improvement, providing a sequence coverage of 80% to 85% for all the charge states of ubiquitin. Overall, this method provides a simple means for the broadband collisional activation of ETnoD ions in the same collision cell in which they are generated for improved structural characterization of polypeptide and protein cations subjected to ETD.     

Electron transfer dissociation versus collisionally activated dissociation of cationized biodegradable polyesters

Biodegradable polyesters were ionized by electrospray ionization and characterized by tandem mass spectrometry using collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) as activation methods. The compounds studied include one homopolymer, polylactide and two copolymers, poly(ethylene adipate) and poly(butylene adipate). CAD of [M+2Na]²⁺ ions from these polyesters proceeds via charge‐remote 1,5‐H rearrangements over the ester groups, leading to cleavages at the (CO)O–alkyl bonds. ETD of the same precursor ions creates a radical anion at the site of electron attachment, which fragments by radical‐induced cleavage of the (CO)O–alkyl bonds and by intramolecular nucleophilic substitution at the (CO)–O bonds. In contrast to CAD, ETD produces fragments in one charge state only and does not cause consecutive fragmentations, which simplifies spectral interpretation and permits conclusive identification of the correct end groups. The radical‐site reactions occurring during ETD are very similar with those reported for ETD of protonated peptides. Unlike multiply protonated species, multiply sodiated precursors form ion pairs (salt bridges) after electron transfer, thereby promoting dissociations via nucleophilic displacement in addition to the radical‐site dissociations typical in ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

Ultraviolet photodissociation of protonated pharmaceuticals in a pressurized linear quadrupole ion trap

Ultraviolet photodissociation (UVPD) was evaluated as a technique for generating ion fragmentation information that is alternative and/or complementary to the information obtained by collision‐induced dissociation (CID). Ions trapped in a pressurized linear ion trap were dissociated using a 355 nm or a 266 nm pulsed laser. Comparisons of UVPD and CID spectra using a set of aromatic chromophore‐containing compounds (desmethyl bosentan, haloperidol, nelfinavir) demonstrated distinct characteristic fragmentation patterns resulting from photodissociation. The wavelength of light and the pressure of the buffer gas in the UVPD cell are important parameters that control fragmentation pathways. The wavelength effect is related to the absorption cross section, location of the chromophore and the energy carried by one photon. Thus, UV irradiation wavelength affects fragmentation pathways as well as the fragmentation rate. The pressure effect can be explained by collisional quenching of ‘slow’ fragmentation pathways. We observed that higher pressure of the buffer gas during UVPD experiments highlights unique fragment ions by suppressing slow fragmentation pathways responsible for CID‐like fragmentation patterns. Copyright © 2010 John Wiley & Sons, Ltd.     

Charge‐Site‐Dependent Dissociation of Hydrogen‐Rich Radical Peptide Cations upon Vacuum UV Photoexcitation

Here, 193 nm vacuum ultraviolet photodissociation (VUVPD) was used to investigate the fragmentation of hydrogen‐rich radical peptide cations generated by electron transfer reactions. VUVPD offers new insight into the factors that drive radical‐ and photon‐directed processes. The location of a basic Arg site influences photon‐activated C_α? C(O) bond cleavages of singly charged peptide radical cations, an outcome attributed to the initial conformation of the peptide as supported by molecular dynamics simulated annealing and the population of excited states upon UV excitation. This hybrid ETD/VUVPD method was employed to identify phosphorylation sites of the kinase domain of human TRPM7/ChaK1.     

Ultraviolet Photodissociation of Carboxylate-Derivatized Peptides in a Quadrupole Ion Trap

The fragmentation patterns obtained by ultraviolet photodissociation (UVPD) and collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer were compared for peptides modified at their C-termini and at acidic amino acids. Attachment of Alexa Fluor 350 or 7-amino-4-methyl-coumarin chromophores at the C-terminal and acidic residues enhances the UV absorptivity of the peptides and all fragment ions that retain the chromophore, such as the y ions that contain the chromophore-modified C-terminus. Whereas CID results in the formation of the typical array of mainly y-type and a/b-type fragment ions, UVPD produces predominantly a/b-type ions with greatly reduced abundances of y ions. Immonium ions, mostly ones from aromatic or basic amino acids, are also observed in the low m/z range upon UVPD. UVPD of peptides containing two chromophore moieties (with one at the C-terminus and another at an acidic residue) results in even more efficient photodissociation at the expense of the annihilation of almost all diagnostic b and y ions containing the chromophore.     

Comparison of CID,ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision‐induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom‐activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b‐/y‐type ions with abundant neutral losses of the phosphorylation modification. ETD produced c‐/z‐type ions in highest abundance but also showed numerous y‐type ions at a frequency about 50% that of the z‐type ions. The major peaks observed in the ETD spectra correspond to the charge‐reduced product ions and small neutral losses from the charge‐reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of glycopeptides by combining collision‐induced dissociation and electron‐transfer dissociation mass spectrometry data

Structural characterization of a glycopeptide is not easily attained through collision‐induced dissociation (CID), due to the extensive fragmentation of glycan moieties and minimal fragmentation of peptide backbones. In this study, we have exploited the potential of electron‐transfer dissociation (ETD) as a complementary approach for peptide fragmentation. Model glycoproteins, including ribonuclease B, fetuin, horseradish peroxidase, and haptoglobin, were used here. In ETD, radical anions transfer an electron to the peptide backbone and induce cleavage of the N–Cα bond. The glycan moiety is retained on the peptide backbone, being largely unaffected by the ETD process. Accordingly, ETD allows not only the identification of the amino acid sequence of a glycopeptide, but also the unambiguous assignment of its glycosylation site. When data acquired from both fragmentation techniques are combined, it is possible to characterize comprehensively the entire glycopeptide. This is being achieved with a mass spectrometer capable of alternating between CID and ETD on‐the‐fly during an LC/MS/MS analysis. This is demonstrated here with several tryptic glycopeptides. Copyright © 2008 John Wiley & Sons, Ltd.     

Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer

Doubly protonated peptides that undergo an electron transfer reaction without dissociation in a linear ion trap can be subjected to beam-type collisional activation upon transfer from the linear ion trap into an adjacent mass analyzer, as demonstrated here with a hybrid triple quadrupole/linear ion trap system. The activation can be promoted by use of a DC offset difference between the ion trap used for reaction and the ion trap into which the products are injected of 12-16 V, which gives rise to energetic collisions between the transferred ions and the collision/bath gas employed in the linear ion trap used for ion/ion reactions. Such a process can be executed routinely on hybrid linear ion trap/triple quadrupole tandem mass spectrometers and is demonstrated here with several model peptides as well as a few dozen tryptic peptides. Collisional activation of the peptide precursor ions that survive electron transfer frequently provides structural information that is absent from the precursor ions that fragment spontaneously upon electron transfer. The degree to which additional structural information is obtained by collisional activation of the surviving singly charged peptide ions depends upon peptide size. Little or no additional structural information is obtained from small peptides (<8 residues) due to the high electron transfer dissociation (ETD) efficiencies noted for these peptides as well as the extensive sequence information that tends to be forthcoming from ETD of such species. Collisional activation of the surviving electron transfer products provided greatest benefit for peptides of 8-15 residues.     

Hypervalent radical formation probed by electron transfer dissociation of zwitterionic tryptophan and tryptophan‐containing dipeptides complexed with Ca2+ and 18‐crown‐6 in the gas phase

The relationship between peptide structure and electron transfer dissociation (ETD) is important for structural analysis by mass spectrometry. In the present study, the formation, structure and reactivity of the reaction intermediate in the ETD process were examined using a quadrupole ion trap mass spectrometer equipped with an electrospray ionization source. ETD product ions of zwitterionic tryptophan (Trp) and Trp‐containing dipeptides (Trp‐Gly and Gly‐Trp) were detected without reionization using non‐covalent analyte complexes with Ca²⁺ and 18‐crown‐6 (18C6). In the collision‐induced dissociation, NH₃ loss was the main dissociation pathway, and loss related to the dissociation of the carboxyl group was not observed. This indicated that Trp and its dipeptides on Ca²⁺(18C6) adopted a zwitterionic structure with an NH₃⁺ group and bonded to Ca²⁺(18C6) through the COO^? group. Hydrogen atom loss observed in the ETD spectra indicated that intermolecular electron transfer from a molecular anion to the NH₃⁺ group formed a hypervalent ammonium radical, R‐NH₃, as a reaction intermediate, which was unstable and dissociated rapidly through N–H bond cleavage. In addition, N–C_α bond cleavage forming the z₁ ion was observed in the ETD spectra of Trp‐GlyCa²⁺(18C6) and Gly‐TrpCa²⁺(18C6). This dissociation was induced by transfer of a hydrogen atom in the cluster formed via an N–H bond cleavage of the hypervalent ammonium radical and was in competition with the hydrogen atom loss. The results showed that a hypervalent radical intermediate, forming a delocalized hydrogen atom, contributes to the backbone cleavages of peptides in ETD. Copyright © 2015 John Wiley & Sons, Ltd.     

Metastable atom‐activated dissociation mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues

Extensive backbone fragmentation resulting in a‐, b‐, c‐, x‐, y‐ and z‐type ions is observed of singly and doubly charged peptide ions through their interaction with a high kinetic energy beam of argon or helium metastable atoms in a modified quadrupole ion trap mass spectrometer. The ability to determine phosphorylation‐sites confirms the observation with previous reports and we report the new ability to distinguish between leucine and isoleucine residues and the ability to cleave two covalent bonds of the proline ring resulting in a‐, b‐, x‐, y‐, z‐ and w‐type ions. The fragmentation spectra indicate that fragmentation occurs through nonergodic radical ion chemistry akin to electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron ionization dissociation mechanisms. However, metastable atom‐activated dissociation mass spectrometry demonstrates three apparent benefits to ECD and ETD: (1) the ability to fragment singly charged precursor ions, (2) the ability to fragment negatively charged ions and (3) the ability to cleave the proline ring that requires the cleavage of two covalent bonds. Helium metastable atoms generated more fragment ions than argon metastable atoms for both substance P and bradykinin regardless of the precursor ion charge state. Reaction times less than 250 ms and efficiencies approaching 5% are compatible with on‐line fragmentation, as would be desirable for bottom‐up proteomics applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Systematic Comparison of Ultraviolet Photodissociation and Electron Transfer Dissociation for Peptide Anion Characterization

Ultraviolet photodissociation at 193?nm (UVPD) and negative electron transfer dissociation (NETD) were compared to establish their utility for characterizing acidic proteomes with respect to sequence coverage distributions (a measure of product ion signals across the peptide backbone), sequence coverage percentages, backbone cleavage preferences, and fragmentation differences relative to precursor charge state. UVPD yielded significantly more diagnostic information compared with NETD for lower charge states (n????2), but both methods were comparable for higher charged species. While UVPD often generated a more heterogeneous array of sequence-specific products (b-, y-, c-, z-, Y-, d-, and w-type ions in addition to a- and x- type ions), NETD usually created simpler sets of a/x-type ions. LC-MS/UVPD and LC-MS/NETD analysis of protein digests utilizing high pH mobile phases coupled with automated database searching via modified versions of the MassMatrix algorithm was undertaken. UVPD generally outperformed NETD in stand-alone searches due to its ability to efficiently sequence both lower and higher charge states with rapid activation times. However, when combined with traditional positive mode CID, both methods yielded complementary information with significantly increased sequence coverage percentages and unique peptide identifications over that of just CID alone.     

Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods

Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y–46 and b+H₂O, are present in PSD, photodissociation, and charge tagging. c^?+57 and z–57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues.

Open image in new window

Graphical Abstract ?

     

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.     

Specific UV photodissociation of tyrosyl-containing peptides in multistage mass spectrometry

UV photodissociation (UVPD) at 262 nm has been carried out on protonated tyrosyl-containing peptides formed by trypsin digestion of apo-transferrin. Under UVPD, the main event is the fragmentation of the C(alpha)-C(beta) bond of the tyrosyl residues leading to a radical ion 107 Da below the precursor ion. The dissociation rate of this specific cleavage appears to be strongly dependent on the peptide sequence and is more prominent on the singly protonated species than on the doubly protonated state. The fragmentation spectra resulting from collisional activation of the protonated even-electron native peptides and of the odd-electron radical species prepared by UVPD are dominated by y-type backbone cleavages. A comparison of their respective y-ion pattern shows complementarities since the combination of both increases the sequence coverage of the peptide sequence. The specific detection of the neutral loss of 107 Da from peptides witnesses the content of at least one tyrosyl residue and, though preliminary, is proposed as a potential new filtering strategy during protein database searching.     

Effects of acidic peptide size and sequence on trivalent praseodymium adduction and electron transfer dissociation mass spectrometry

Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M + Pr ‐ H]²⁺; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues generate [M + Pr]³⁺, which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M + Pr + H]⁴⁺; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non‐metallated c‐ and z‐ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N‐terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal‐peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III). Copyright © 2017 John Wiley & Sons, Ltd.     

Ultraviolet photodissociation of chromophore-labeled oligosaccharides via reductive amination and hydrazide conjugation

The fragmentation patterns of hydrazide-conjugated and reductively aminated oligosaccharides, including lacto-N-fucopentaoses and lacto-N-difucohexaoses, produced on collisionally induced dissociation (CID) and ultraviolet photodissociation (UVPD) in a quadrupole ion trap are presented. The two derivatization methods generate different cross-ring cleavages on UVPD and CID. UVPD of hydrazide-conjugated oligosaccharides yield predominant (2, 4)A-type cross-ring cleavage ions. In contrast, UVPD of aminated oligosaccharides results mainly in (0, 1)A-type ions. Moreover, more extensive dual-cleavage pathways (i.e. internal fragment ions) were observed on UVPD.