Single series peptide fragment ion spectra generated by two-stage collision-induced dissociation in a triple quadrupole

By using nanoelectrospray ionization and a triple quadrupole analyzer, simplified fragment ion spectra of peptides have been recorded by combining skimmer collision-induced dissociation with precursor ion scanning or neutral loss scanning. These pseudo-MS³ scan modes are characterized by two-stage collision-induced dissociation and have been termed sCID/precursor and sCID/neutral loss scan, respectively. By these scan modes, peptide fragment ion spectra can be generated that predominantly show signals of a single fragment ion series, such as the B or Y″ series. Skimmer collision-induced dissociation combined with scanning for neutral loss of 28 generates spectra showing B ions, whereas combination with precursor ion scanning for the Y″₁ ion results in spectra showing Y″ ions for tryptic peptides (Y″₁=m/z 147 for C-terminal lysine, Y″₁=m/z 175 for C-terminal arginine). Sequence information including the direction of the sequence is easily extracted from the simplified fragment ion spectra generated by two-stage collision-induced dissociation, because the scan mode defines the type of fragments observed. The analytical results reported are similar to those that have been achieved in MS³ experiments using a hybrid BEQQ or a pentaquadrupole mass spectrometer (Schey, K. L.; Schwartz, J. C.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1989, 3, 305–309). The pseudo-MS³ technique used in this study has some limitations with respect to sample purity, because there is no step of mass selection before the first stage of collisional activation; however, it has the advantage that a standard triple quadrupole instrumentation can be used.     

Charge-remote fragmentation of taurine-conjugated bile acids

Taurine-conjugated bile acids are an important group of biological metabolites. When investigated by negative-ion fast-atom bombardment collision-induced dissociation mass spectroscopy they show charge-remote fragmentations of the [M-H]- pseudomolecular ion. These fragmentations provide information on the positions of ring substituents remote from the charge site. In the present work we have compared the negative-ion fast-atom bombardment collision-induced dissociation spectra of six different conjugated bile acids.     

Mechanism of cross-ring cleavage reactions in dirhamnosyl lipids: effect of the alkali ion

Liquid secondary ion mass spectrometry and high-energy collision-induced dissociation were used to analyze a dirhamnosyl lipid mixture. The negative fast-atom bombardment spectrum reveals a mixture of four homologous dirhamnosyl lipids with the following general structure: Rha-Rha-Cn-Cm (where Cn and Cm denote 3-hydroxy fatty acid moieties). The mass region 450-600 u in the collision-induced dissociation spectra of the negative [M - H]- ions shows product ions that can be rationalized by terminal loss of a 3-hydroxyalkanoic acid residue; these ions can be used for the characterization of the fatty acid substituents. A unique effect of alkali-metal ions on the course of fragmentation of dirhamnosyl lipid attachment ions was observed. The strong chelation of sodium is revealed from the stability of the [M - H + 2Na]+ ion that does not lose a sodium ion with the eliminated neutrals, contrary to what is observed for the dilithium adduct. Cross-ring cleavages occur during high-energy collision-induced dissociation of both positively and negatively charged precursor ions. The results suggest a concerted decomposition pathway involving the six-membered rings of the monosaccharide residues. The formation of cross-ring cleavage products, which retain the C10-C10 moiety during high-energy collision-induced dissociation of all the precursor ions that contain sodium or lithium, strongly supports a retro [2 + 2 + 2] mechanism.     

Determination of the glycosylation site in flavonoid mono-O-glycosides by collision-induced dissociation of electrospray-generated deprotonated and sodiated molecules

The influence of the glycosylation site on the fragmentation behavior of 18 flavonoid glycoside standards was studied using positive and negative electrospray ionization mass spectrometry in combination with collision-induced dissociation and tandem mass spectrometry. The glycosylation position is shown to affect the relative abundance of the radical aglycone ions that can be observed in the [M-H]- collision-induced dissociation spectra. In particular, the radical aglycone ions are very abundant for deprotonated flavonol 3-O-glycosides. Collisional activation of the radical aglycone ions produced from positional isomers revealed minor differences: m,nB0- product ions are pronounced for 7-O-glycosides, whereas m,nA0- product ions are relatively more abundant for 4'-O-glycosides. In addition, the ratio between the radical aglycone and the regular aglycone ions in the [M+Na]+ high-energy collision-induced dissociation spectra gives an indication about the glycosylation site. This ion ratio allows the differentiation between flavonoid 3-O- and 7-O-glycosides or can be useful in the comparison of unknown compounds with standards. Unambiguous differentiation between O-glycosylation at the common positions of flavonoid O-glycosides, i.e. the 3-, 4'- and 7-positions, is achieved by collisional activation of sodiated molecules at high collision energy. The presence of a B-ring product ion containing the sugar residue indicates 4'-O-glycosylation, whereas the loss of the B-ring part from the aglycone product ion is characteristic of 3-O-glycosylation and the loss of the B-ring part from both the [M+Na]+ precursor ion and the aglycone product ion points to 7-O-glycosylation.     

Chiral discrimination of alpha-amino acids by the DNA triplet GCA

The DNA triplet GCA is successfully used for the first time as a chiral selector for the chiral discrimination and optical purity measurement of some alpha-amino acids by investigating the collision-induced dissociation spectra of the sodiated ternary complex ion formed by electrospray ionization.     

A study of the gas-phase reaction between protonated acetaldehyde and methanol

Protonated acetaldehyde is methylated on the oxygen during interaction with methanol in the gas phase. The ionic product of the ion/molecule reaction between methanol and protonated acetaldehyde is identical with C-protonated methylvinyl ether (high-pressure ionization), and with the (M − C₂H₅)⁺ fragment ion of sec-butyl methyl ether (following electron ionization), and also with the (M − OCH₃)⁺ fragment ion of acetaldehyde dimethylacetal (following electron ionization). The structures of these ions and the mechanism of their formation were established by isotope-labeling experiments and collision-induced dissociation mass spectra of model compounds obtained with three different types of tandem mass spectrometers (BEQQ, triple-quadrupole, and a penta-quadrupole instrument). Gas phase synthesis of the product ion from [²H₃]-methanol or [²H₄]-acetaldehyde provided insight into its mode of formation and collision-induced dissociation.     

Grazing incidence surface-induced dissociation of protonated peptides generated by matrix-assisted laser desorption/ionization

The grazing incidence surface-induced dissociation (GI-SID) of various protonated peptides with typical kinetic energies of 350 eV was investigated. Peptide ions were generated by matrix-assisted laser desorption/ionization (MALDI) using delayed extraction. The collision target surfaces used were aluminum and a liquid film of perfluorinated hydrocarbons. All peptides studied in these experiments showed enhanced fragment ion yields at grazing incidence (GI-SID effect) as observed in our former experiments with other precursor ion types. In general the GI-SID spectra exhibit N-terminal a(1)-type fragment ions, immonium ions and side-chain fragment ions in the low mass-to-charge region. Fragment ion series of the peptide backbone were not observed, which are typical and abundant in the spectra of established fragmentation techniques like collision-induced dissociation, MALDI post-source decay or surface-induced dissociation at steeper angles. The potential of the GI-SID process to yield useful information for primary structure determination of peptides is indicated by the observed differences in the GI-SID spectra of the isomeric dipeptides LR and IR.     

Modification of a double focusing mass spectrometer for the observation of metastable ions decaying in two field-free regions

The occurrence of ‘metastable’ ions formed in the ion source as one species decays to a lower mass ion prior to mass analysis provides direct evidence for dissociation mechanisms in organic mass spectra. Both unimolecular and collision-induced decay processes provide useful information on available dissociation pathways, ion structures, and energetics. In double-focusing mass spectrom-eters the existence of two field-free regions presents a convenient vehicle for studying these processes and for obtaining kinetic information on consecutive metastables through simple modification in circuitry and operating conditions. These modifications are described and the method is used to demonstrate consecutive metastable decay processes in acetyl acetone, 1-butene, toluene and alumin-um hexafluoroacetylacetonate. Drift tube pressure studies showed that, within experimental error, consecutive metastables observed in acetyl acetone and 1-butene were collision-induced, while toluene and the aluminum chelate both exhibit two-step unimolecular decays.     

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.     

Electron impact-induced fragmentation of 2,1-benzisothiazoline 2,2-dioxide

The electron impact-induced fragmentation patterns of 2,1-benzisothiazoline 2,2-dioxide nitro derivatives were studied. The rationalizations proposed for the fragmentations are supported by accurate mass measurements, daughter ion (mass analysed ion kinetic energy and B/E linked-scan), parent ion, and constant neutral loss spectra in metastable and collision-induced dissociation modes and deuterium labelling.     

Electrospray ionization and tandem mass spectrometry of cysteinyl eicosanoids: Leukotriene C4 and FOG7

The cysteinyl leukotrienes, LTC4, LTD4 and LTE4, and the recently described cysteinyl eicosanoid, 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) have been analyzed by tandem mass spectrometry. Both [M-H]- and [M+H]+ ions were produced by electrospray ionization and collision-induced dissociation of these molecular ion species were studied using both an ion trap and a triple quadrupole instrument. Product ion spectra obtained were characteristic of the structure of the cysteinyl leukotrienes and mechanisms of ion formation were investigated by using deuterium-labeled analogs. The product ion spectrum obtained following collision-induced dissociation of the [M-H]- anion from FOG7 was devoid of significant structural information and further studies of collision activation of the [M+H]+ spectrum were therefore examined. Positive ion MS3 spectra obtained in the ion trap from the gamma-glutamate cleavage products of FOG7 and its derivative (d7-FOG7) afforded an abundant ion not observed in spectra generated from the cysteinyl leukotrienes. Formation of this fragment ion likely occurred via a McLafferty-type rearrangement to afford cleavage of the C6-C7 bond adjacent to the sulfur atom and was valuable for the identification of the structure of FOG7 and defining the biosynthetic pathway as a 1,4-Michael addition of glutathione to 5-oxo-eicosatetraenoic acid (5-oxo-ETE).     

两种氨基酸中[MH-CO2H2]+的特征质谱碎裂

运用低能碰撞诱导解离（ＣＩＤ）研究了电子轰击（ＥＩ）、快原子轰击（ＦＡＢ）电离条件下质子化亮氨酸与异亮氨酸解离产生亚稳离子「ＭＨ－ＣＯ２Ｈ２」的单分子质谱破裂，二种异构体呈现出了各自不同的解离特征，根据ＣＩＤ的特征碎片离子和氘代同位素标记实验，提出了其破裂过程的存在离子／中性（碎片）复合物中间体机理，并对有关的特征离子的形成进行了讨论。     

The use of static pressures of heavy gases within a quadrupole ion trap

The performance of quadrupole ion traps using argon or air as the buffer gas was evaluated and compared to the standard helium only operation. In all cases a pure buffer gas, not mixtures of gases, was investigated. Experiments were performed on a Bruker Esquire ion trap, a Finnigan LCQ, and a Finnigan ITMS for comparison. The heavier gases were found to have some advantages, particularly in the areas of sensitivity and collision-induced dissociation efficiency; however, there is a significant resolution loss due to dissociation and/or scattering of ions. Additionally, the heavier gases were found to affect ion activation and deactivation during MS/MS, influencing the product ion intensities observed. Finally, the specific quadrupole ion trap design and the ion ejection parameters were found to be crucial in the quality of the spectra obtained in the presence of heavy gases. Operation with static pressures of heavy gases can be beneficial under certain design and operating conditions of the quadrupole ion trap.     

Ion structure determination through collisions of ions of high kinetic energy

Through studies on [C₆H₆]+ and [C₆H₅]+ it is shown that charge stripping provides information on ion structure which is complementary to that given by collision-induced dissociation. Stripping involves a more uniform sampling over the range of ion internal energies than does collision-induced dissociation which preferentially samples higher energy ions.     

An unprecedented rearrangement in collision-induced mass spectrometric fragmentation of protonated benzylamines

The collision-induced dissociation (CID) mass spectra of several protonated benzylamines are described and mechanistically rationalized. Under collision-induced decomposition conditions, protonated dibenzylamine, for example, loses ammonia, thereby forming an ion of m/z 181. Deuterium labeling experiments confirmed that the additional proton transferred to the nitrogen atom during this loss of ammonia comes from the ortho positions of the phenyl rings and not from the benzylic methylene groups. A mechanism based on an initial elongation of a C--N bond at the charge center that eventually cleaves the C--N bond to form an ion/neutral complex of benzyl cation and benzylamine is proposed to rationalize the results. The complex then proceeds to dissociate in several different ways: (1) a direct dissociation to yield a benzyl cation observed at m/z 91; (2) an electrophilic attack by the benzyl cation within the complex on the phenyl ring of the benzylamine to remove a pair of electrons from the aromatic sextet to form an arenium ion, which either donates a ring proton (or deuteron when present) to the amino group forming a protonated amine, which undergoes a charge-driven heterolytic cleavage to eliminate ammonia (or benzylamine) forming a benzylbenzyl cation observed at m/z 181, or undergoes a charge-driven heterolytic cleavage to eliminate diphenylmethane and an immonium ion; and (3) a hydride abstraction from a methylene group of the neutral benzylamine to the benzylic cation to eliminate toluene and form a substituted immonium ion. Corresponding benzylamine and dibenzylamine losses observed in the spectra of protonated tribenzylamine and tetrabenzyl ammonium ion, respectively, indicate that the postulated mechanism can be widely applied. The postulated mechanisms enabled proper prediction of mass spectral fragments expected from protonated butenafine, an antifungal drug.     

Positive-ion mass spectra and collision-induced dissociation of some 2,3-didehydro amino acids

The positive-ion mass spectra of a number of didehydro amino acids, ionized by electron impact and/or thermospray, and collision-induced dissociation spectra taken at collision energies of a few electron volts and keV have been performed on multiple quadrupole and reversed geometry sector instruments. Observed differences in the mass spectra and in the fragmentation patterns are explained in terms of different isomeric structures, different internal excitation energies and different ion transit times between the ion source and the collision cell. Molecular ions of unhydrated amino acids are efficiently formed both by electron impact and thermospray, whilst molecular ions of the hydrated compounds are formed more efficiently by the latter technique. The present investigation demonstrates that the use of different ionization techniques combined with mass spectrometry/mass spectrometry measurements at different collision energies yields a wealth of information relevant to structural characterization of this important class of molecules.     

Stereospecific fragmentation of starfish polyhydroxysteroids in electrospray ionization mass spectrometry

Electrospray ionization mass spectra and collision-induced dissociation mass spectra in positive and negative ion modes of five polyhydroxysteroid compounds from starfish were studied. Tandem mass spectra exhibit extensive fragmentation, including sequential neutral losses of H₂O molecules and cleavages in the tetracyclic nucleus and side chains. The relative intensity of some peaks in tandem mass spectra enables stereoisomers with the different orientations of the hydroxyl group at C15 in the tetracyclic nucleus to be distinguished. Some data on the fragmentation mechanisms were obtained by H–D exchange and mass spectrometry analysis.     

Trimethylsilyl group migration in the mass spectra of trimethylsilyl ethers of cholesterol oxidation products. product ion characterization by linked-scan tandem mass spectrometry

Loss of the A ring in the electron-impact mass spectra of the trimethylsilyl (TMS) derivatives of several cholesterol oxidation products is accompanied by intramolecular migration of the 3-O-TMS group to the charge-retaining portion of the molecule. Linked-scan techniques (B/E and B²/E) were used to establish the fragmentation processes leading to the formation of the rearrangement ion. The TMS group appears to migrate to heteroatomic sites in the 5-; 6-, or 7-positions of the B ring. This structural assignment is supported by isotopic labeling studies and collision-induced dissociation of the resulting product ion.     

Half protons or doubly charged protons? The history of metastable ions

The history of metastable ions is intertwined with that of ions formed by collision-induced dissociation (CID), and frequently the genesis of the two ion types cannot be strictly separated. Originally, metastable ions were considered a curiosity or even a nuisance, being responsible for "humps" in the base line of the recorded mass spectra. In their heyday metastable ions were recognized as having importance for establishing fragmentation sequences and for distinguishing between isomeric ion structures. Today, in many respects the utility of metastable ions has been superseded by a systematic application of CID techniques; yet the evaluation of their shape is still of importance for questions of reaction energies and ion thermochemistry.     

2,5—双（4—羟基苯亚甲基）环戊酮中双电荷离子存在的串联质谱证据

应用串联质谱的碰撞诱导解离和联动扫描技术，研究了２，５－双（４－羟基苯亚甲基）环戊酮的质谱解离特征，提供了双电荷离子存在的实验证据。进一步对双电荷离子（ｍ／ｚ１４６）的碰撞诱导解离碎裂进行了讨论。