The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Doubly charged porphyrin ion tandem mass spectrometry: Implications for structure elucidation

Under electron ionization (EI) conditions, porphyrins yield unusually high intensities of doubly charged molecular and fragment ions. These doubly charged ions offer unique opportunities for the structure elucidation of porphyrins by tandem mass spectrometry (MS/MS). First, they fragment to a greater extent than the corresponding singly charged ions under both EI/MS and EI/MS/MS conditions. Second, doubly and singly charged porphyrin ions often fragment via different pathways, and can therefore yield different structural information. This paper describes several ways in which analyses of doubly charged porphyrin ions with a triple quadrupole tandem mass spectrometer can be useful in structure elucidation of porphyrins. The effect of the metal atom on the fragmentation of metalloporphyrins in an EI source is demonstrated by correlating the extent of doubly charged fragment ion formation to a stability index. Doubly charged porphyrin ions are shown to yield predominantly doubly charged daughter ions upon collisionally activated dissociation (CAD), and are also shown to fragment to a greater extent than corresponding singly charged porphyrin ions. Advantages and disadvantages of doubly charged porphyrin ion MS/MS for structure elucidation are discussed.     

Structural determination of the novel fragmentation routes of zwitteronic morphine opiate antagonists naloxonazine and naloxone hydrochlorides using electrospray ionization tandem mass spectrometry

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of the zwitteronic salts naloxonazine dihydrochloride 1 and naloxone hydrochloride 2, a common series of morphine opiate receptor antagonists, were recorded using different declustering potentials. The singly charged ion [M+H-2HCl](+) at m/z 651.3170 and the doubly charged ion [M+2H-2HCl](2+) at m/z 326.1700 were noted for naloxonazine dihydrochloride 1; and the singly charged ion [M+H-HCl](+) at m/z 328.1541 was observed for naloxone hydrochloride 2. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) experiments established the fragmentation routes of these compounds. In addition to the characteristic diagnostic product ions obtained, we noticed the formation of a series of radical product ions for the zwitteronic compounds 1 and 2, and also the formation of a distonic ion product formed from the singly charged ion [M+H-HCl](+) of naloxone hydrochloride 2. Confirmation of the various established fragmentation routes was effected by conducting a series of ESI-CID-QqTof-MS/MS product ion scans, which were initiated by CID in the atmospheric pressure/vacuum interface using a higher declustering potential. Deuterium labeling was also performed on the zwitteronic salts 1 and 2, in which the hydrogen atoms of the OH and NH groups were exchanged with deuterium atoms. Low-energy CID-QqTof-MS/MS product ion scans of the singly charged and doubly charged deuteriated molecules confirmed the initial fragmentation patterns proposed for the protonated molecules. Precursor ion scan analyses were also performed with a conventional quadrupole-hexapole-quadrupole tandem mass spectrometer and allowed the confirmation of the genesis of some diagnostic ions.     

Sequence information, distinction and quantitation of C-terminal leucine and isoleucine in ternary complexes of tripeptides with Cu(II) and 2,2'-bipyridine

Tripeptides form ternary complexes with Cu(2+) and 2,2'-bipyridine (bpy) that self-assemble upon mixing the components in aqueous methanol solution. Electrospray ionization (ESI) of the complex solutions provides abundant singly charged [Cu(peptide -- H)bpy](+) and doubly charged [Cu(peptide)bpy](2+) ions. Collision-induced dissociation (CID) at low ion kinetic energies of several tripeptides, AGG, GGA, LGG, GGL, GGI, FGG, GGF, LGF, GLF, GFL, GYA and GAY, showed fragments that were indicative of the amino acid sequence in the peptide. In addition, CID of single and doubly charged complexes of isomeric tripeptides GGL and GGI provided unambiguous distinction of the isomeric leucine and isoleucine residues. Leucine peptides eliminated C(3)H(7) radicals from the amino acid side-chain whereas isoleucine eliminated C(2)H(5) radicals. CID of gas-phase doubly charged peptide complexes in a quadrupole ion trap produced a series of singly charged sequence fragments that following isolation and further CID furnished distinct fragments that allowed quantitation of leucine and isoleucine-containing peptides in mixtures.     

Structural characterization of polyethers using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Fragmentation of polyethers, such as poly(ethylene glycol) (PEG), poly(propylene glycol) and poly(tetramethylene glycol) was analyzed by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) using a quadrupole ion trap time-of-flight mass spectrometer (QIT-ToF). The Li adduct ion provided more abundant fragments than the Na and K adduct ions in the MS/MS spectra. A previous study had demonstrated four series fragments of hydroxyl-, vinyl- and formyl-terminated ions, as well as distonic cations, in high-energy fast atom bombardment MS/MS and MALDI collision-induced dissociation measurements of poly(ethylene glycol). In the present study, the low-energy MS/MS measurements using MALDI-QIT-ToF, showed hydroxyl-, vinyl- and formyl- terminated fragments with or without other fragment groups, but not distonic cations. The fragmentation depended on the types of polyethers examined. MS/MS measurements using MALDI-QIT-ToF are expected to allow structural characterization of unknown components of polyethers.     

Mutual storage mode ion/ion reactions in a hybrid linear ion trap

Ion/ion proton transfer reactions involving mutual storage of both ion polarities in a linear ion trap (LIT) that comprises part of a hybrid triple quadrupole/linear ion trap mass spectrometer have been effected. Mutual ion storage in the x- and y-dimensions arises from the normal operation of the oscillating quadrupole field of the quadrupole array, while storage in the z-dimension is enabled by applying unbalanced radio-frequency amplitudes to opposing sets of rods of the array. Efficient trapping (>90%) is achieved for thermalized ions over periods of several seconds. Reactions were demonstrated for multiply charged protein/peptide cations formed by electrospray with anions derived from glow discharge ionization of perfluoro(methyldecalin) (PMD) introduced from the side of the LIT rod array. Doubly and singly charged protein ions are readily formed via ion/ion reactions. The parameters that affect ion/ion reactions are discussed, including the degree of RF unbalance on the LIT rods, vacuum pressure, nature of the buffer gas, reaction time, anion abundance, and the low mass cutoff for ion/ion reaction. The present system has a demonstrated upper mass-to-charge ratio limit of at least 33,000. The system also has high flexibility with respect to defining MS(n) experiments involving both collision-induced dissociation (CID) and ion/ion reactions. Experiments are demonstrated involving beam-type CID in the pressurized collision quadrupole (Q2) followed by ion/ion reactions involving the product ions in the LIT. Ion parking experiments are also demonstrated using the mutual storage ion/ion reaction mode in the LIT, with a parking efficiency over 60%.     

Use of doubly protonated molecules in the analysis of cathedulins in crude extracts of khat (Catha edulis) by liquid chromatography/serial mass spectrometry

Analysis of crude methanolic extracts of fresh khat (Catha edulis) by liquid chromatography/mass spectrometry (LC/MS) revealed the presence of 62 cathedulin alkaloids (compared with 15 published structures). Many cathedulins generated doubly protonated molecules following electrospray ionisation and the ratio of doubly to singly protonated species could be manipulated by adjusting the electrospray capillary position and source conditions. By selecting the doubly protonated species for serial mass spectrometric analysis (MS/MS), it was possible to use an ion trap mass spectrometer to observe singly charged product ions at lower m/z values than ion trap MS/MS analysis of [M+H](+) would have allowed. These spectra were particularly valuable in elucidating the acylation patterns of cathedulins where MS/MS analysis of [M+H](+) resulted in loss of a large neutral species to yield a small singly charged fragment below the lower limit for ion trapping. Acylation patterns for most of the 62 cathedulins are proposed from mass spectrometric analysis, and the data obtained for a major unreported cathedulin of mass 1001 Da suggest that it belongs to a new group of cathedulins having a cathate dilactone bridge but not an evoninate bridge.     

Dissociation of polyether-transition metal ion dimer complexes in a quadrupole ion trap

The formation and dissociation of dimer complexes consisting of a transition metal ion and two polyether ligands is examined in a quadrupole ion trap mass spectrometer. Reactions of three transition metals (Ni, Cu, Co) with three crown ethers and four acyclic ethers (glymes) are studied. Singly charged species are created from ion-molecule reactions between laser-desorbed monopositive metal ions and the neutral polyethers. Doubly charged complexes are generated from electrospray ionization of solutions containing metal salts and polyethers. For the singly charged complexes, the capability for dimer formation by the ethers is dependent on the number of available coordination sites on the ligand and its ability to fully coordinate the metal ion. For example, 18-crown-6 never forms dimer complexes, but 12-crown-4 readily forms dimers. For the more flexible acyclic ethers, the ligands that have four or more oxygen atoms do not form dimer complexes because the acyclic ligands have sufficient flexibility to wrap around the metal ion and prevent attachment of a second ligand. For the doubly charged complexes, dimers are observed for all of the crown ethers and glymes, thus showing no dependence on the flexibility or number of coordination sites of the polyether. The nonselectivity of dimer formation is attributed to the higher charge density of the doubly charged metal center, resulting in stronger coordination abilities. Collisionally activated dissociation is used to evaluate the structures of the metal-polyether dimer complexes. Radical fragmentation processes are observed for some of the singly charged dimer complexes because these pathways allow the monopositive metal ion to attain a more favorable 2 + oxidation state. These radical losses are observed for the dimer complexes but not for the monomer complexes because the dimer structures have two independent ligands, a feature that enhances the coordination geometry of the complex and allows more flexibility for the rearrangements necessary for loss of radical species. Dissociation of the doubly charged complexes generated by electrospray ionization does not result in losses of radical neutrals because the metal ions already exist in favorable 2+ oxidation states.     

Use of Doubly Charged Precursors to Validate Dissociation Mechanisms of Singly Charged Poly(Dimethylsiloxane) Oligomers

Collision-induced dissociation of doubly charged poly(dimethylsiloxane) (PDMS) molecules was investigated to provide experimental evidence for fragmentation reactions proposed to occur upon activation of singly charged oligomers. This study focuses on two PDMS species holding trimethylsilyl or methoxy end-groups and cationized with ammonium. In both cases, introduction of the additional charge did not induce significant differences in dissociation behavior, and the use of doubly charged precursors enabled the occurrence of charge-separation reactions, allowing molecules always eliminated as neutrals upon activation of singly charged oligomers to be detected as cationized species. In the case of trimethylsilyl-terminated oligomers, random location of the adducted charge combined with rapid consecutive reactions proposed to occur from singly charged precursors could be validated based on MS/MS data of doubly charged oligomers. In the case of methoxy-terminated PDMS, favored interaction of the adducted ammonium with both end-groups, proposed to rationalize the dissociation behavior of singly charged molecules, was also supported by MS/MS data obtained for molecules adducted with two ammonium cations.

High amplitude short time excitation: A method to form and detect low mass product ions in a quadrupole ion trap mass spectrometer

Collision induced dissociation (CID) in a quadrupole ion trap mass spectrometer using the conventional 30 ms activation time is compared with high amplitude short time excitation (HASTE) CID using 2 ms and 1 ms activation times. As a result of the shorter activation times, dissociation of the parent ions using the HASTE CID technique requires resonance excitation voltages greater than conventional CID. After activation, the rf trapping voltage is lowered to allow product ions below the low mass cut-off to be trapped. The HASTE CID spectra are notably different from those obtained using conventional CID and can include product ions below the low mass cut-off for the parent ions of interest. The MS/MS efficiencies of HASTE CID are not significantly different when compared with the conventional 30 ms CID. Similar results were obtained with a two-dimensional (linear) ion trap and a three-dimensional ion trap.     

Scanning a triple quadrupole mass spectrometer for doubly charged ions

Charge exchange reactions within a triple quadrupole mass spectrometer characterize doubly charged ions formed in the ion source. Two methods have been developed for identifying the singly charged ions formed from doubly charged ions by charge exchange in the collision quadrupole. The first is based on the characteristically high kinetic energy-to-charge ratios of the products of charge exchange; this property can be used to separate these ions from all other singly charged ions. This retarding potential method is analogous to procedures for recording doubly charged ion mass spectra using sector instruments. The second method is based on the fact that, although mass remains constant in the charge exchange reaction, the change in mass-to-charge ratio can be followed. A charge exchange linked scan, predicated on changes in charge rather than mass, but otherwise analogous to neutral loss/gain scans, is described. Information on the structure of doubly charged ions can be obtained by recording the fragmentation products of dissociative charge exchange. The utility of the charge exchange linked scan for the selective identification of polynuclear aromatic compounds in a complex mixture is described. The methods given can be generalized to cover other charge permutation reactions.     

Unimolecular and collision-induced reactions of doubly charged porphyrins

Results from a tandem mass spectrometry (MS/MS) study, obtained with a reverse-geometry mass spectrometer, of the unimolecular and collision-induced reactions of doubly charged free-base and metal containing alkyl-substituted porphyrins formed by electron ionization are reported. These doubly charged porphyrin ions dissociate to yield both singly and doubly charged product ions via a number of reactions. This article classifies the major reactions observed, illustrating each with the appropriate spectra. Supplementary data from the same porphyrins, acquired with a tandem quadrupole MS/MS instrument, are also presented. The potential utility of some of these reactions as new methods for porphyrin analysis is discussed.     

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.     

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.     

Elucidation of high micro-heterogeneity of an acidic-neutral trichotoxin mixture from Trichoderma harzianum by electrospray ionization quadrupole time-of-flight mass spectrometry

The high micro-heterogeneity of an acidic-neutral trichotoxin mixture from T. harzianum, PC01, was elucidated using a modern tandem mass spectrometer equipped with an electrospray ionization source, a hybrid quadrupole-orthogonal accelerator and a reflectron time-of-flight analyzer. The trichotoxins appeared predominantly in six possible doubly charged pseudo molecular ions with three different adducts (H, Na and K) as [M + 2H](2+), [M + H + Na](2+), [M + H + K](2+), [M + 2Na](2+), [M + Na + K](2+) and [M + 2K](2+). The singly charged pseudomolecular ions, [M + H](+), [M + Na](+) and [M + K](+), occurred only in low abundance when the cone voltages were higher than 30 V. Additional singly charged fragments, b(12) and y"6 (complementary N- and C-terminal fragments), were obtained in high abundance using high cone voltages. The peak patterns of both singly and doubly charged molecular adducts revealed that this trichotoxin mixture contained several components having 6-7 molecular masses with a consecutive 14 u difference among members in the same molecular adduct series. Furthermore, well resolved isotopic peaks of every doubly or singly charged ions and their reproducible peak intensity allowed the identification of the mixing of acidic trichotoxins 1 u molecular mass heavier than the neutral counterparts in the sample. Tandem mass spectrometric (MS/MS) analyses of various singly charged b(12) and y"6 ions supported the sequence deduction of the major and minor components and also the position of Glu in the sequences of these acidic molecules. The setting of either low or high resolution of the quadrupole mass filter unit together with a suitable variation of the collision voltage for any MS/MS precursor were the tools for extracting a number of mixed components and obtaining the major and minor sequences of these precursor peaks. The nature of the MS/MS fragmentation and the data assignment of three major doubly charged ions, [M + 2H](2+), [M + K + H](2+) and [M + 2K](2+), are demonstrated. Eleven new sequences of both acidic and neutral trichotoxins are reported.     

Capillary electrophoresis-electrospray mass spectra of the herbicides paraquat and diquat

The positive ion electrospray mass spectra of the quaternary ammonium salt herbicides paraquat and diquat are examined by on-line separation with capillary electrophoresis (CE) and by direct infusion of the analytes. The analytes are separated by CE in 7–10 min at pH 3.9 in 50% methanol-water by using several different separation buffer electrolytes. The capillary electrophoresis-electrospray ionization (CE-ES) mass spectra of paraquat and diquat consist primarily of doubly charged molecular ions, singly charged molecular ions, and singly charged deprotonated ions. The direct infusion spectra consist primarily of doubly charged molecular ions and singly charged deprotonated ions. The relative abundances of the doubly charged and deprotonated ions depend strongly on the presence or absence of ammonium ion in the CE separation buffer or the direct infusion solution. A deprotonation mechanism is proposed in which the free base ammonia is the deprotonating agent in the desolvating charged droplets or in the gas phase. The analytical potential of the CE-ES electrospray approach for environmental analyses is evaluated in terms of the precision of replicate injections, linear concentration range, and estimated detection limit.     

一些双电荷离子的气相碎裂反应

借助质量分析离子动能谱和串联质谱研究了由电子轰击产生的双电荷离子的单分子亚稳碎裂及碰撞诱导分解过程,讨论了两种实验方法导致的差别因素.此外,根据质量分析离子动能谱提供的双电荷离子电荷分离反应的动能释放值计算了两电荷中心间距的最小值,以判别按不同电荷分离方式碎裂的双电荷离子的过渡态结构.     

UV photodissociation of phospho-seryl-containing peptides: laser stabilization of the phospho-seryl bond with multistage mass spectrometry

Protonated precursor ions of phosphorylated peptides containing a tyrosyl residue have been subjected to UV laser-induced dissociation (LID) at a wavelength of 220 nm and to collision-induced dissociation (CID) in an ion trap. As expected, neutral loss of the phosphate group is one of the predominant fragmentation channels during CID together with H2O elimination. In contrast, LID leads mainly to the homolytic cleavage of the tyrosyl side chain and a restrained loss of the phosphate group. Interestingly, the intensity of the dephosphorylated fragment ion is greatly minimized when CID is carried out next on the radical precursor ion of the singly and doubly charged species.     

Generation and collision-induced dissociation of ammonium tetrafluoroborate cluster ions

Singly and doubly charged cluster ions of ammonium tetrafluoroborate (NH4BF4) with general formula [(NH4BF4)nNH4]+ and [(NH4BF4)m(NH4)2]2+, respectively, were generated by electrospray ionization (ESI) and their fragmentation examined using collision-induced dissociation (CID) and ion-trap tandem mass spectrometry. CID of [(NH4BF4)nNH4]+ caused the loss of one or more neutral NH4BF4 units. The n = 2 cluster, [(NH4BF4)2NH4]+, was unique in that it also exhibited a dissociation pathway in which HBF4 was eliminated to create [(NH4BF4)(NH3)NH4]+. Dissociation of [(NH4BF4)m(NH4)2]2+ occurred through two general pathways: (a) 'fission' to produce singly charged cluster ions and (b) elimination of one or more neutral NH4BF4 units to leave doubly charged product ions. CID profiles, and measurements of changing precursor and product ion signal intensity as a function of applied collision voltage, were collected for [(NH4BF4)nNH4]+ and compared with those for analogous [(NaBF4)nNa]+ and [(KBF4)nK]+ ions to determine the influence of the cation on the relative stability of cluster ions. In general, the [(NH4BF4)nNH4]+ clusters were found to be easier to dissociate than both the sodium and potassium clusters of comparable size, with [(KBF4)nK]+ ions the most difficult to dissociate.     

Multi-stage tandem mass spectrometry of metal cationized leucine enkephalin and leucine enkephalin amide

We have examined the multi-stage collision induced dissociation (CID) of metal cationized leucine enkephalin, leucine enkephalin amide, and the N-acetylated versions of the peptides using ion trap mass spectrometry. In accord with earlier studies, the most prominent species observed during the multi-stage CID of alkali metal cationized leucine enkephalin are the [b(n) + 17 + Cat]+ ions. At higher CID stages (i.e. >MS(4)), however, dissociation of the [b2 + 17 + Cat]+ ion, a cationized dipeptide, results in the production of [a(n) -1 + Cat]+ species. The multi-stage CID of Ag+ cationized leucine enkephalin can be initiated with either the [b(n) -1 + Ag]+ or [b(n) + 17 + Ag]+ ions produced at the MS/MS stage. For the former, sequential CID stages cause, in general, the loss of CO, and then the loss of the imine of the C-terminal amino acid, to reveal the amino acid sequence. Similar to the alkali cationized species, CID of [b2 -1 + Ag]+ produces prominent [a(n) -1 + Ag]+ ions. The multi-stage CID of argentinated peptides is reminiscent of fragmentation observed for protonated peptides, in that a series of (b(n)) and (a(n)) type ions are generated in sequential CID stages. The Ag+ cation is similar to the alkali metals, however, in that the [b(n) + 17 + Ag]+ product is produced at the MS/MS and MS3 stages, and that sequential CID stages cause the elimination of amino acid residues primarily from the C-terminus. We found that N-acetylation of the peptide significantly influenced the fragmentation pathways observed, in particular by promoting the formation of more easily interpreted (in the context of unambiguous sequence determination) dissociation spectra from the [b2 + 17 + Li]+, [b2 + 17 + Na]+ and [b2 -1 + Ag]+ precursor ions. Our results suggest, therefore, that N-acetylation may improve the efficacy of multi-stage CID experiments for C-terminal peptide sequencing in the gas phase. For leucine enkephalin amide, only the multi-stage CID of the argentinated peptide allowed the complete amino acid sequence to be determined from the C-terminal side.