The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions

The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions (generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’ this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly from the mass-to-charge ratio assignments of the CID spectrum.     

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.     

On performing simultaneous electron transfer dissociation and collision-induced dissociation on multiply protonated peptides in a linear ion trap

We propose a tandem mass spectrometry method that combines electron-transfer dissociation (ETD) with simultaneous collision-induced dissociation (CID), termed ETD/CID. This technique can provide more complete sequence coverage of peptide ions, especially those at lower charge states. A selected precursor ion is isolated and subjected to ETD. At the same time, a residual precursor ion is subjected to activation via CID. The specific residual precursor ion selected for activation will depend upon the charge state and m/z of the ETD precursor ion. Residual precursor ions, which include unreacted precursor ions and charge-reduced precursor ions (either by electron-transfer or proton transfer), are often abundant remainders in ETD-only reactions. Preliminary results demonstrate that during an ETD/CID experiment, b, y, c, and z-type ions can be produced in a single experiment and displayed in a single mass spectrum. While some peptides, especially doubly protonated ones, do not fragment well by ETD, ETD/CID alleviates this problem by acting in at least one of three ways: (1) the number of ETD fragment ions are enhanced by CID of residual precursor ions, (2) both ETD and CID-derived fragments are produced, or (3) predominantly CID-derived fragments are produced with little or no improvement in ETD-derived fragment ions. Two interesting scenarios are presented that display the flexibility of the ETD/CID method. For example, smaller peptides that show little response to ETD are fragmented preferentially by CID during the ETD/CID experiment. Conversely, larger peptides with higher charge states are fragmented primarily via ETD. Hence, ETD/CID appears to rely upon the fundamental reactivity of the analyte cations to provide the best fragmentation without implementing any additional logic or MS/MS experiments. In addition to the ETD/CID experiments, we describe a novel dual source interface for providing front-end ETD capabilities on a linear ion trap mass spectrometer.     

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.     

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.     

苯二酚异构体双电荷离子和单电荷离子的动能谱研究

本文利用质量分析离子动能谱(MIKES)、碰撞诱导解离(CID)技术和电子捕获诱导解离(ECID)技术, 研究了邻、间、对苯二酚分子在电子轰击质谱(EIMS)中产生的双电荷离子[C6H6O2]^2^+, [C6H4O]^2^+和单电荷离子[C6H6O2]^+。根据测定的电荷分离反应动能释放值T和由此计算出的两电荷间距R, 推测出过渡态的结构。有趣的是, 可利用单电荷离子[C6H6O2]^+的MIKES/CID谱区分苯二酚异构体。     

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.     

A study of the electron capture induced decompositions and charge separation reactions of the doubly charged isomeric ions of the methylpyridines and aniline

The doubly charged isomeric ions [C₆H₇N]²⁺ formed from 2-, 3- and 4-methylpyridine and aniline were investigated via their unimolecular charge separation reactions and by electron capture induced decompositions (ECID). The ECID spectra were compared with the collision induced decomposition (CID) spectra of the singly charged ions in an attempt to investigate the structure of the doubly charged ions. The four isomers could be unambiguously identified by their unimolecular charge separations. These differences were greater than in the mass spectra, ECID spectra or CID spectra of singly charged ions.     

Strategies for generating peptide radical cations via ion/ion reactions

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon–iodine (C―I) bond are subjected to UVPD with 266‐nm photons, which selectively cleaves the C―I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even‐electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non‐covalent complexes in the electrospray process. Copyright © 2015 John Wiley & Sons, Ltd.     

Electrospray Ionization Tandem Mass Spectrometry of Ammonium Cationized Polyethers

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine’s degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.     

Ion mobility augments the utility of mass spectrometry in the identification of human hemoglobin variants

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.     

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.     

Two ion/ion charge inversion steps to form a doubly protonated peptide from a singly protonated peptide in the gas phase

An approach is described to increase the degree of protonation of a polypeptide ion in the gas phase. Sequential charge inversion reactions involving the reactions of oppositely charged ions are used to yield a net increase in ion charge. The approach is illustrated here with the conversion of singly protonated bradykinin to doubly protonated bradykinin. The first step involves conversion of the singly protonated peptide to the singly deprotonated peptide via reactions with multiply charged anions derived from carboxylate-terminated dendrimers. Some of the singly deprotonated peptide was then converted to doubly protonated peptide via reactions with multiply charged cations derived from amino-terminated dendrimers. The overall approach is illustrative of a general strategy for increasing the absolute charge states of large ions in the gas phase.     

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.     

Protein identification via surface-induced dissociation in an FT-ICR mass spectrometer and a patchwork sequencing approach

Surface-induced dissociation (SID) and collision-induced dissociation (CID) are ion activation techniques based on energetic collisions with a surface or gas molecule, respectively. One noticeable difference between CID and SID is that SID does not require a collision gas for ion activation and is, therefore, directly compatible with the high vacuum requirement of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Eliminating the introduction of collision gas into the ICR cell for collisional activation dramatically shortens the acquisition time for MS/MS experiments, suggesting that SID could be utilized for high-throughput MS/MS studies in FT-ICR MS. We demonstrate for the first time the utility of SID combined with FT-ICR MS for protein identification. Tryptic digests of standard proteins were analyzed using a hybrid 6-tesla FT-ICR mass spectrometer with SID and CID capabilities. SID spectra of mass-selected singly and doubly charged peptides were obtained using a diamond-coated target mounted at the rear trapping plate of the ICR cell. The broad internal energy distribution deposited into the precursor ion following collision with the diamond surface allowed a variety of fragmentation channels to be accessed by SID. Composition and sequence qualifiers produced by SID of tryptic peptides were used to improve the statistical significance of database searches. Protein identification MASCOT scores obtained using SID were comparable or better than scores obtained using sustained off-resonance irradiation collision-induced dissociation (SORI-CID), the conventional ion activation technique in FT-ICR MS.     

Tandem mass spectrometric analysis of (13)C-containing ions from a mixture of homologous peptides differing by one mass unit at a residue

Tandem mass spectrometry of a mixture of two peptides that differ from each other by a single mass unit due to mutation is presented. The mutant beta-globin of hemoglobin Hoshida is present along with the normal counterpart, and the amino acid substitution of glutamine for glutamic acid is located within tryptic peptide T5 of M(r) 2057. 9. The mass of the mutated peptide is 1 u lower. In the isotopic cluster for the doubly charged ion of the peptide T5, the resolved ion with mass of 1030.0 represents the normal peptide with 93 (12)C atoms and the mutated one with 92 (12)C and one (13)C atoms. Collision-induced dissociation (CID) of this composite ion identified the mutation by presenting a key fragment derived from the (12)C-only mutant peptide, as reported in a previous study. Similarly, when an ion containing multiple (13)C atoms was selected as a precursor for CID, the mutation could be identified, even in large fragments, by a marked change in the shape of the isotopic cluster for the consecutive product ions. This study demonstrates the merit of selecting a resolved ion rather than the whole isotopic cluster as a precursor in the CID measurements of large peptides or proteins for characterizing heterozygous mutations.     

Comparison of infrared multiphoton dissociation and collision-induced dissociation of supercharged peptides in ion traps

The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision-induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with m-nitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared with that obtained by CID for all charge states investigated. The number of diagnostic ions increased with the charge state for IRMPD; however, this trend was not consistent for CID because the supercharged ions did not always yield the greatest number of diagnostic ions. Significantly different fragmentation pathways were observed for the different charge states upon CID or IRMPD with the latter yielding far more immonium ions and often fewer uninformative ammonia, water, and phosphoric acid neutral losses. Pulsed-Q dissociation resulted in an increase in the number of internal product ions, a decrease in sequence-informative ions, and reduced overall ion abundances. The enhanced sequence coverage afforded by IRMPD of supercharged ions was demonstrated for a variety of model peptides, as well as for a tryptic digest of cytochrome c.     

Comparison of collision-induced dissociation and electron-induced dissociation of <Emphasis Type="Italic">singly protonated</Emphasis> aromatic amino acids,cystine and related simple peptides using a hybrid linear ion trap–FT-ICR mass spectrometer

The gas-phase fragmentation reactions of singly protonated aromatic amino acids, their simple peptides as well as simple models for intermolecular disulfide bonds have been examined using a commercially available hybrid linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Low-energy collision-induced dissociation (CID) reactions within the linear ion trap are compared with electron-induced dissociation (EID) reactions within the FT-ICR cell. Dramatic differences are observed between low-energy CID (which occurs via vibrational excitation) and EID. For example, the aromatic amino acids mainly fragment via competitive losses of NH(3) and (H(2)O+CO) under CID conditions, while side-chain benzyl cations are major fragment ions under EID conditions. EID also appears to be superior in cleaving the S-S and S-C bonds of models of peptides containing an intermolecular disulfide bond. Systematic studies involving fragmentation as a function of electron energy reveal that the fragmentation efficiency for EID occurs at high electron energy (more than 10 eV) compared with the low-electron energy (less than 0.2 eV) typically observed for electron capture dissociation fragmentation. Finally, owing to similarities between the types of fragment ions observed under EID conditions and those previously reported in ultraviolet photodissociation experiments and the electron-ionization mass spectra, we propose that EID results in fragmentation via electronic excitation and vibrational excitation. EID may find applications in analyzing singly charged molecular ions formed by matrix-assisted laser desorption ionization.