Metal ion adducts in the structural analysis of ginsenosides by electrospray ionization with multi-stage mass spectrometry

The effect of metal (Li+, Na+, K+, Ag+) cationization on collision-induced dissociation of ginsenosides was investigated by electrospray ionization mass spectrometry combined with multi-stage mass spectrometry (ESI-MS(n)). The fragments of sodiated and lithiated molecules give valuable structural information regarding the nature of the aglycone and the sequence and linkage information of sugar moieties. However, the number and relative abundances of fragment ions from lithiated ginsenosides are significantly greater than for the sodiated species. The K+ adducts undergo glycosidic cleavages and very limited cross-ring reactions. The silver ion adducts fragment mainly through glycosidic cleavages.     

Characterization of iridoids by fast atom bombardment mass spectrometry followed by collision-induced dissociation of

Fast atom bombardment mass spectra of a series of naturally occurring and synthetically modified iridoid glycosides were studied using lithium cationization and collision-induced dissociation of the resulting [M+Li]+ ions. Lithium cationization leads to the unambiguous determination of the molecular masses of these compounds. Collision-induced dissociation of the lithiated molecular ions give valuable structural information regarding the nature of the substituent on both the aglycone and the sugar moieties. The characteristic fragmentation pathways identified are (1) elimination of neutral molecules comprising the substituents on either the aglycone or sugar moieties, (2) formation of lithiated aglycone and their fragment ions, (3) formation of lithiated sugar and their fragment ions, (4) fragmentation corresponding to the cleavage of the aglycone or sugar ring and (5) fragmentation characteristic of the substituents present in either the aglycone or sugar parts of the molecule. Elimination of two acyloxy radicals from the lithiated molecular ion is a characteristic fragmentation in the case of acyloxy derivatives.     

Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.     

Investigation of double-stranded DNA/drug interaction by ESI/FT ICR: orientation of dissociations relates to stabilizing salt bridges

Noncovalent complexes of DNA and Hoechst 33258 were investigated by ESI-FT/ICR MS in various activation modes (collision-induced dissociation (CID), sustained off-resonance irradiation collision-induced dissociation (SORI-CID), infrared multiphoton dissociation (IRMPD) and electron detachment dissociation (EDD)). The binding selectivity of Hoechst 33258 was confirmed by the comparative study of its noncovalent association with different DNA sequences. The CID spectra of [ds + HO - 5H](5-) obtained with a linear hexapole ion trap resulted in unzipping of the strands. This outcome is a clue to the drug-binding mode, shading light on the localization of the binding sites of Hoechst 33258 to the DNA sequence. The IRMPD and SORI-CID experiments mainly gave DNA backbone cleavages and internal fragment ions. From this result, information on the localization of the binding sites of Hoechst 33258 in the DNA sequence was obtained. No sodium cationization was observed on the DNA sequence ions although they were present on fragmentation of the duplex, indicating that the backbone cleavages were generated from the single strand associated with the Hoechst 33258 where the number of alkali cation is restricted. Under electron detachment (ED) conditions, multiple EDs were achieved for the [ds + HO - 5H](5-) ion without any significant dissociation. The presence of drug appears to enhance the stability of the multiply charged system. It was proposed that the studied noncovalent complex involved the formation of zwitterions and consequently strong salt-bridge interactions between DNA and drug.     

Effects of transition metal ion coordination on the collision-induced dissociation of polyalanines

Transition metal-polyalanine complexes were analyzed in a high-capacity quadrupole ion trap after electrospray ionization. Polyalanines have no polar amino acid side chains to coordinate metal ions, thus allowing the effects metal ion interaction with the peptide backbone to be explored. Positive mode mass spectra produced from peptides mixed with salts of the first row transition metals Cr(III), Fe(II), Fe(III), Co(II), Ni(II), Cu(I), and Cu(II) yield singly and doubly charged metallated ions. These precursor ions undergo collision-induced dissociation (CID) to give almost exclusively metallated N-terminal product ions whose types and relative abundances depend on the identity of the transition metal. For example, Cr(III)-cationized peptides yield CID spectra that are complex and have several neutral losses, whereas Fe(III)-cationized peptides dissociate to give intense non-metallated products. The addition of Cu(II) shows the most promise for sequencing. Spectra obtained from the CID of singly and doubly charged Cu-heptaalanine ions, [M + Cu - H](+) and [M + Cu](2+) , are complimentary and together provide cleavage at every residue and no neutral losses. (This contrasts with [M + H](+) of heptaalanine, where CID does not provide backbone ions to sequence the first three residues.) Transition metal cationization produces abundant metallated a-ions by CID, unlike protonated peptides that produce primarily b- and y-ions. The prominence of metallated a-ions is interesting because they do not always form from b-ions. Tandem mass spectrometry on metallated (Met = metal) a- and b-ions indicate that [b(n) + Met - H](2+) lose CO to form [a(n) + Met - H](2+), mimicking protonated structures. In contrast, [a(n) + Met - H](2+) eliminate an amino acid residue to form [a(n-1) + Met - H](2+), which may be useful in sequencing.     

Non-covalent interactions of alkali metal cations with singly charged tryptic peptides

The complexes formed by alkali metal cations (Cat(+) = Li(+), Na(+), K(+), Rb(+)) and singly charged tryptic peptides were investigated by combining results from the low-energy collision-induced dissociation (CID) and ion mobility experiments with molecular dynamics and density functional theory calculations. The structure and reactivity of [M + H + Cat](2+) tryptic peptides is greatly influenced by charge repulsion as well as the ability of the peptide to solvate charge points. Charge separation between fragment ions occurs upon dissociation, i.e. b ions tend to be alkali metal cationised while y ions are protonated, suggesting the location of the cation towards the peptide N-terminus. The low-energy dissociation channels were found to be strongly dependant on the cation size. Complexes containing smaller cations (Li(+) or Na(+)) dissociate predominantly by sequence-specific cleavages, whereas the main process for complexes containing larger cations (Rb(+)) is cation expulsion and formation of [M + H](+). The obtained structural data might suggest a relationship between the peptide primary structure and the nature of the cation coordination shell. Peptides with a significant number of side chain carbonyl oxygens provide good charge solvation without the need for involving peptide bond carbonyl groups and thus forming a tight globular structure. However, due to the lack of the conformational flexibility which would allow effective solvation of both charges (the cation and the proton) peptides with seven or less amino acids are unable to form sufficiently abundant [M + H + Cat](2+) ion. Finally, the fact that [M + H + Cat](2+) peptides dissociate similarly as [M + H](+) (via sequence-specific cleavages, however, with the additional formation of alkali metal cationised b ions) offers a way for generating the low-energy CID spectra of 'singly charged' tryptic peptides.     

Electron capture dissociation and infrared multiphoton dissociation of oligodeoxynucleotide dications

We report electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) of doubly protonated and protonated/alkali metal ionized oligodeoxynucleotides. Mass spectra following ECD of the homodeoxynucleotides polydC, polydG, and polydA contain w or d "sequence" ions. For polydC and polydA, the observed fragments are even-electron ions, whereas radical w/d ions are observed for polydG. Base loss is seen for polydG and polydA but is a minor fragmentation pathway in ECD of polydC. We also observe fragment ions corresponding to w/d plus water in the spectra of polydC and d(GCATGC). Although the structure of these ions is not clear, they are suggested to proceed through a pentavalent phosphorane intermediate. The major fragment in ECD of d(GCATGC) is a d ion. Radical a- or z-type fragment ions are observed in most cases. IRMPD primarily results in base loss, but backbone fragmentation is also observed. IRMPD provides more sequence information than ECD, but the spectra are more complex due to extensive base and water losses. It is proposed that the smaller degree of sequence coverage in ECD, with fragmentation mostly occurring close to the ends of the molecules, is a consequence of a mechanism in which the electron is captured at a P=O bond, resulting in a negatively charged phosphate group. Consequently, at least two protons (or alkali metal cations) must be present to observe a w or d fragment ion, a requirement that is less likely for small fragments.     

Infrared multiphoton dissociation of small-interfering RNA anions and cations

Infrared multiphoton dissociation (IRMPD) on a linear ion trap mass spectrometer is applied for the sequencing of small interfering RNA (siRNA). Both single-strand siRNAs and duplex siRNA were characterized by IRMPD, and the results were compared with that obtained by traditional ion trap-based collision induced dissociation (CID). The single-strand siRNA anions were observed to dissociate via cleavage of the 5′ P—O bonds yielding c- and y-type product ions as well as undergo neutral base loss. Full sequence coverage of the siRNA anions was obtained by both IRMPD and CID. While the CID mass spectra were dominated by base loss ions, accounting for ∼25% to 40% of the product ion current, these ions were eliminated through secondary dissociation by increasing the irradiation time in the IRMPD mass spectra to produce higher abundances of informative sequence ions. With longer irradiation times, however, internal ions corresponding to cleavage of two 5′ P—O bonds began to populate the product ion mass spectra as well as higher abundances of [a − Base] and w-type ions. IRMPD of siRNA cations predominantly produced c- and y-type ions with minimal contributions of [a − Base] and w-type ions to the product ion current; the presence of only two complementary series of product ions in the IRMPD mass spectra simplified spectral interpretation. In addition, IRMPD produced high abundances of protonated nucleobases, [G + H]⁺, [A + H]⁺, and [C + H]⁺, which were not detected in the CID mass spectra due to the low-mass cut-off associated with conventional CID in ion traps. CID and IRMPD using short irradiation times of duplex siRNA resulted in strand separation, similar to the dissociation trends observed for duplex DNA. With longer irradiation times, however, the individual single-strands underwent secondary dissociation to yield informative sequence ions not obtained by CID.     

Study of the dissociation of a charge-reduced phosphopeptide formed by electron transfer from an alkali metal target

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.     

Lithium and transition metal ions enable low energy collision-induced dissociation of polyglycols in electrospray ionization mass spectrometry

Electrospray ionization tandem mass spectrometry has the potential to be widely used as a tool for polymer structural characterization. However, the backbones or molecular chains of many industrial polymers including functional polyglycols are often difficult to dissociate in tandem mass spectrometers using low energy collision-induced dissociation (CID). We present a method that uses Li+ and transition metal ions such as Ag+ as the cationization reagents for electrospray ionization in an ion trap mass spectrometer. It is shown that lithium and transition metal polyglycol adduct ions can be readily fragmented with low energy CID. Comparative results from different cationization reagents in their abilities of producing both MS spectra and CID spectra are shown. This method opens the possibility of using conventional and readily available low energy CID tandem MS to study polyglycol structures.     

Tandem mass spectra of ammonium ion,metal ion and ligated metal ion adducts of acyclic sugar derivatives

The tandem mass (MS/MS) spectra of ammonium ion, metal ion and ligated metal ion adducts of chain-extended acyclic nitro-containing deoxyglucose and deoxygalactose derivatives have been studied. The ammonium adducts fragment primarily by elimination of ammonia followed by acetic acid, thus not giving much structural information. In contrast, cationization of these compounds by metal ions and ligated metal ions gave structurally informative and useful fragment ions on MS/MS. The metal ions and ligated metal ions play an important role in controlling and directing fragmentation. Retro-aldol fragmentation is facilitated by metal ions such as Li(+), Na(+), Ag(+) and Cu(+), whereas the adducts with higher alkali metal ions such as Rb(+) and Cs(+) fragment to give only the corresponding metal ions. The divalent metal ions such as Cu(2+) and Ba(2+) also induce retro-aldol fragmentation. However, the charge is carried by the aldehyde fragment in the case of Cu(2+) adducts, whereas the nitroalkane fragment carries the charge in the case of Ba(2+) adducts. Ligated metal ions such as ZnCl(+), CuCl(+), InCl(2) (+) and BaCl(+) also behave similarly and induce retro-aldol fragmentation in these acyclic sugars. Both the metal ion and ligated metal ion adducts can fragment by elimination of metal-containing neutral molecules.     

Electron Transfer Dissociation of Milk Oligosaccharides

For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.     

Effect of alkali metal cationization and multiple alkali metal exchange on the collision-induced dissociation of loganic acid studied by electrospray ionization tandem mass spectrometry

Under electrospray ionization conditions loganic acid undergoes alkali metal (Li, Na and K) exchange and alkali metal cationization. Multiple exchanges of up to four alkali metal ions are observed. Different populations of metal exchanged species are produced during electrospray ionization. Collision-induced dissociation of ammonium cationized species is compared with that of metal cationized species to study the effect of metal cationization. Glycosidic cleavage and ring cleavages of aglycone and sugar moieties are the major fragmentation pathways observed during collision-induced dissociation. The fragmentations of the highly metal exchanged species indicate the opening of the pyran ring. Collision-induced dissociation of the various metal exchanged and metal cationized species also reveals the nature of the different populations. Copyright 2000 John Wiley & Sons, Ltd.     

MS<Superscript>n</Superscript> characterization of protonated cyclic peptides and metal complexes

MSⁿ experiments involving low energy collisionally activated dissociation (CAD) in a quadrupole ion trap were used to characterize the fragmentation of alkali, alkaline earth and transition metal complexes of five cyclic peptides, and the results were compared with those obtained for protonated cyclic peptides. Complexes with metal ions produced enhanced abundances of the most diagnostic fragments for elucidating the primary structures. For cyclosporin A, nickel and lithium complexes gave additional sequence information compared with the protonated peptide. For depsipeptides, sodium and lead complexes were superior to the protonated peptide or other metal complexes for sequencing residues, and CAD of the lead complexes led to preferential cleavage of two residues at a time. For cyclic lipopeptides, complexes with silver, nickel and strontium ions provided enhanced abundances of key fragment ions.     

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.     

High-energy collision-induced dissociation of small polycyclic aromatic hydrocarbons

High-energy collision-induced dissociation (CID) experiments on polycyclic aromatic hydrocarbons (PAHs) having 2-6 rings, naphthalene, anthracene, phenanthrene, fluoranthene, pyrene and coronene, were performed, and the relative abundances of their fragment ions were investigated as a function of collision energy. The results revealed that the PAHs except naphthalene showed a bimodal-type distribution of positive fragmentation ions, which is closely similar to the fragment-ion distribution reported for the CID of three-dimensional fullerene, C(60)(+) and C(70)(+). The three-ring isomers of anthracene and phenanthrene and the four-ring isomers of fluoranthene and pyrene can be distinguishable in their spectra under an electron ionization energy of 70 eV, but the high-energy CID spectra of the three- and four-ring isomers were almost identical. The fragmentation corresponding to fragment ions in the low-mass region of the bimodal CID spectra could be interpreted by the simple statistical model that fragment ions are formed by random evaporation from the molecular ions after a considerable structural rearrangement, 'phase transition', occurring at some high-energy state.     

Mass spectral study of alkali-cationized Boc-carbo-beta3-peptides by electrospray tandem mass spectrometry

Electrospray tandem mass spectrometry was used to study the dissociation reactions of [M+Cat]+ (Cat = Na+ and Li+) of Boc-carbo-beta3-peptides. The collision-induced dissociation (CID) spectra of [M+Cat-Boc]+ of these peptides are found to be significantly different from those of [M+H-Boc]+ ions. The spectra are more informative and display both C- and N-terminus metallated ions in addition to characteristic fragment ions of the carbohydrate moiety. Based on the fragmentations observed in the CID spectra of the [M+Cat-Boc]+ ions, it is suggested that the dissociation involves complexes in which the metal ion is coordinated in a multidentate arrangement involving the carbonyl oxygen atoms. The CID spectra of [M+Cat-Boc]+ ions of the peptide acids show an abundant N-terminal rearrangement ion [b(n)+17+Cat]+ which is absent for esters. Further, two pairs of positionally isomeric Boc-carbo-beta3-peptide acids, Boc-NH-Caa(S)-beta-hGly-OH (11) and Boc-NH-beta-hGly-Caa(S)-OH (12), and [Boc-NH-Caa(S)-beta-hGly-Caa(S)-beta-hGly-OH] (13) and [Boc-NH-beta-hGly-Caa(S)-beta-hGly-Caa(S)-OH] (14), were differentiated by the CID of [M+Cat-Boc]+ ions. The CID spectra of compounds 11 and 13 are significantly different from those of 12 and 14, respectively. The abundance of [b(n)+17+Cat]+ ions is higher for peptide acids 12 and 14 with a sugar group at the C-terminus when compared to 11 and 13 which contain a sugar moiety at the N-terminus. The observed differences between the CID spectra of these isomeric peptides are attributed to the difference in the preferential site of metal ion binding and also on the structure of the cyclic intermediate involved in the formation of the rearrangement ion.     

Protonating polymer oligomers in the gas phase to change fragmentation pathways

Ionization of polymers in mass spectrometry is usually achieved by forming metal ion adducts. The metal ion has been shown by Wesdemiotis to often play a spectator role in the collision-induced dissociation (CID) chemistry of these species, wherein they fragment according to a free-radical mechanism similar to that found in their pyrolysis. The result is a predominance of low-mass ions in the CID mass spectrum. We have changed this behavior by generating protonated oligomers in the gas phase by first forming proton-bound complexes of the oligomers with amino acids or peptides by electrospray ionization. These complexes dissociate first by loss of the amino acid/peptide to form protonated oligomers, which then undergo a unique fragmentation chemistry. In this article we discuss the results for poly(methyl methacrylate) (PMMA) and poly(butyl acrylate) (PBA). Initially, protonated PMMA and PBA lose methanol and butanol, respectively, from the side chains of the respective monomers. The resulting PMMA-derived ion then undergoes a series of neutral losses corresponding to 32 and 28 Da, methanol and carbon monoxide. This continues as collision energy increases until a final, carbon-rich backbone ion is formed, which then undergoes a classic hydrocarbon fragmentation pattern. The PBA-derived ions are proposed to fragment by the loss of butylether molecules to form anhydride rings along the oligomer chain. The number of ether molecules lost corresponded to half the number of available side chains in the oligomer. The resulting poly-anhydride ion dissociates by small molecule loss. Mechanisms have been suggested for the fragmentation chemistry of these two classes of oligomers.     

High energy collision-induced dissociation of alkali-metal ion adducts of crown ethers and acyclic analogs.

High energy collision-induced dissociation (CID) techniques were applied for structural elucidation of alkali-metal ion adducts of crown ethers. The CID of alkali-metal adducts of tetraglyme and hexaethylene glycol were also evaluated to contrast the fragmentation pathways of the cyclic ethers with those of acyclic analogs. A common fragmentation channel for alkali-metal ion adducts of all the ethers, which results in distonic radical cations, is the homolytic cleavage of carbon-carbon bonds. Additionally, dissociation by carbon-oxygen bond cleavages occurs, and these processes are analogous to the fragmentation pathways observed for simple protonated ethers. The proposed fragmentation pathways for alkali-metal ion adducts of crown ethers result mostly in odd-electron, acyclic product ions. Dissociation of the alkali-metal ion adducts of the acyclic ethers is dominated by losses of various neutral species after an initial hydride or proton transfer. The CID processes for all ethers are independent of the alkali-metal ion sizes; however, the extent of dissociation of the complexes to bare alkali-metal ions increases with the size of the metal.     

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.