Selective detection of phosphopeptides in complex mixtures by electrospray liquid chromatography/mass spectrometry

A mass spectrometry-based method that does not involve the use of radiolabeling was developed for selective detection of phosphopeptides in complex mixtures. Mixtures of phosphorylated and nonphosphorylated peptides at the low picomole level are analyzed by negative ion electrospray liquid chromatography/mass spectrometry using C-18 packed fused-silica columns (≤320-μm i.d.). Peptides and phosphopeptides in the chromatographic eluant undergo collision-induced dissociation in the free-jet expansion region prior to the mass analyzing quadrupole. Using relatively high collisional excitation potentials, phospho|peptides containing phosphoserine, phosphothreonine, and phosphotyrosine fragment to yield diagnostic ions at m/z 63 and 79 corresponding to PO₂ ^?; and PO₃ ^?, respectively. Chromatographic peaks containing phosphopeptides are indicated where these diagnostic ions maximize. The highest sensitivity for phosphopeptide detection is obtained using selected-ion monitoring for m/z 63 and 79. Full-scan mass spectra that exhibit the diagnostic phosphopeptide fragment ions, together with pseudomolecular ions, may be obtained by stepping the collisional excitation potential from a high value during the portion of each scan in which the low-mass-to-charge ratio diagnostic marker ions are being detected to a lower value while the upper mass-to-charge ratio range is being scanned. Good sensitivity for phosphopeptide detection was achieved using standard trifluoroacetic acid containing mobile phases for reversed-phase high-performance liquid chromatography. Data illustrating the selectivity and sensitivity of the approach are presented for mixtures of peptides and phosphopeptides containing the three commonly phosphorylated amino acids.     

Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode

A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.     

Losses of ions during forward and reverse scans in a quadrupole ion trap mass spectrometer and how to reduce them

The higher order fields present in the quadrupole ion trap may have beneficial effects such as increases in mass resolution in the mass-selective instability or resonance ejection modes of operation, but may also result in losses of ions due to nonlinear resonances. In this work, the reduction in ion intensities observed in the mass spectra of polyethylene glycol (PEG 1000) has been utilized to monitor the ion losses resulting from these higher order fields during the rf voltage scans in both the forward and reverse directions. Extensive ion losses were observed in reverse rf voltage scans at q _z=0.64 (a _z=0), which corresponds to octopole resonance at β _z=1/2. The losses depended upon rf voltage scan rate and ion mass being greater for lower scan rates and lower masses. For ions of m/z 877, losses of up to 60% of the stored ions were observed at low scan rates (<1×10⁴ Da/s), but were minimal at higher scan rates. Thus, it is possible to avoid such losses during reverse scans by scanning the region q _z=0.64 at rates in excess of 4×10⁴ Da/s. In forward rf voltage scans, ion storage was considerably more reliable, with significant losses observed only at very high scan rates near the region q _z=0.78 (hexapole resonance at β _z=2/3).     

Selective detection of the tolyl cation among other [C7H7]+ isomers by ion/molecule reaction with dimethyl ether

The ion/molecule reaction of the tolyl cation with dimethyl ether has been investigated using triple quadrupole mass spectrometry. Three isomers with [C₇H₇]⁺ composition, the 3-tolyl, benzyl, and tropylium cations, were individually selected and reacted with dimethyl ether at a pressure of 1 mtorr in the second quadrupole (Q₂) collision cell. Only the tolyl ion reacted to yield a methoxylated product ion peak at m/z 122. This reaction product having m/z 122 is postulated to be identical in structure with the molecular ion of 3-methyl anisole, as supported by thermochemical data and the similarity of the collision induced dissociation (CID) daughter ion mass spectra of the product ion and the molecular ion of authentic 3-methyl anisole. The daughter ion mass spectra of the three [C₇H₇]⁺ isomers during CID, by using a triple quadrupole mass spectrometer, are nearly identical; on the other hand, the analytical approach based on the ion/molecule reaction with dimethyl ether clearly exhibits distinct gas-phase chemistry reflecting structural differences among the isomers. Sot     

Calibration Point for Electron Ionization MS / MS spectra measured with multiquadrupole mass spectrometers

A protocol for establishing standard instrument conditions for measurement of product ion MS/MS spectra from parent ions produced by electron ionization is presented. Within this protocol, the ion at m/z 231 (C₅F₉ ⁺) from perfluorokerosene or perfluorotributylamine is selected as the parent ion and subjected to collision-induced dissociation. The relative intensities of product ions at m/z 69, 131, and 181 are monitored as a function of collision energy while keeping the target gas pressure constant within the range of 10^?4–10^?6 torr (measured), or a beam attenuation of approximately 30-70%. The collision energy at which the ion intensities for product ions at m/z 69 and 181 are equal is defined as the calibration point at that collision gas pressure; the intensity of the ion at m/z 131 is very close to this value as well. Electron ionization MS/MS spectra taken at the calibration point using two different multiquadrupole instruments show good reproducibility for several test compounds. The high degree of similarity may aid in the establishment of a MS/MS spectral library.     

A Distonic Radical-Ion for Detection of Traces of Adventitious Molecular Oxygen (O2) in Collision Gases Used in Tandem Mass Spectrometers

We describe a diagnostic ion that enables rapid semiquantitative evaluation of the degree of oxygen contamination in the collision gases used in tandem mass spectrometers. Upon collision-induced dissociation (CID), the m/z 359 positive ion generated from the analgesic etoricoxib undergoes a facile loss of a methyl sulfone radical [^?SO₂(CH₃); 79-Da] to produce a distonic radical cation of m/z 280. The product-ion spectrum of this m/z 280 ion, recorded under low-energy activation on tandem-in-space QqQ or QqTof mass spectrometers using nitrogen from a generator as the collision gas, or tandem-in-time ion-trap (LCQ, LTQ) mass spectrometers using purified helium as the buffer gas, showed two unexpected peaks at m/z 312 and 295. This enigmatic m/z 312 ion, which bears a mass-to-charge ratio higher than that of the precursor ion, represented an addition of molecular oxygen (O₂) to the precursor ion. The exceptional affinity of the m/z 280 radical cation towards oxygen was deployed to develop a method to determine the oxygen content in collision gases. Figure

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Protonated Dipeptide Losses from b 5 and b 4 Ions of Side Chain Hydroxyl Group Containing Pentapeptides

In this study, C-terminal protonated dipeptide eliminations were reported for both b ₅ and b ₄ ions of side chain hydroxyl group (–OH) containing pentapeptides. The study utilized the model C-terminal amidated pentapeptides having sequences of XGGFL and AXVYI, where X represents serine (S), threonine (T), glutamic acid (E), aspartic acid (D), or tyrosine (Y) residue. Upon low-energy collision-induced dissociation (CID) of XGGFL (where X?=?S, T, E, D, and Y) model peptide series, the ions at m/z 279 and 223 were observed as common fragments in all b ₅ and b ₄ ion (except b ₄ ion of YGGFL) mass spectra, respectively. By contrast, peptides, namely S_MeGGFL-NH₂ and E_OMeGGFL-NH₂, did not show either the ion at m/z 279 or the ion at m/z 223. It is shown that the side chain hydroxyl group is required for the possible mechanism to take place that furnishes the protonated dipeptide loss from b ₅ and b ₄ ions. In addition, the ions at m/z 295 and 281 were detected as common fragments in all b ₅ and b ₄ ion (except b ₄ ion of AYVYI) mass spectra, respectively, for AXVYI model peptide series. The MS⁴ experiments exhibited that the fragment ions at m/z 279, 223, 295, and 281 entirely reflect the same fragmentation behavior of [M?+?H]⁺ ion generated from commercial dipeptides FL-OH, GF-OH, YI-OH, and VY-OH. These novel eliminations reported here for b ₅ and b ₄ ions can be useful in assigning the correct and reliable peptide sequences for high-throughput proteomic studies.

Individual b2 ion fragmentation profiles combined with AspN digestion improve N-terminal peptide sequencing

The N terminus of peptides generated by AspN is restricted to about 40 dipeptide motifs starting with D or E. These motifs are visible upon collision-induced dissociation (CID) as b₂ ions, which are often the most abundant low-mass fragment ions. It was observed that b₂ ions are accompanied by a set of sequence-specific neutral losses of CO, H₂O, NH₃, and some other small units. To test the utility of these profiles as additional parameters for reliable assignment of the b₂ ion motif besides its m/z value, the CID spectra of 221 different AspN-generated peptides covering all N-terminal D-X and E-X motifs were recorded. Qualitatively, the b₂ ion fragmentation profiles of individual motifs were found to exhibit little dependency on the rest of the peptide sequence. Thus, it is concluded that the set of b₂ ion fragmentation profiles recorded in this study can be used as reference set. Knowledge of these profiles provides an increased specificity for b₂ ion annotation of AspN-generated peptides compared to the use of only a solitary b₂ ion m/z value. Recognition of the b₂ ion motif provides a two-amino-acid sequence including its direction; it provides the location of this motif at the N terminus, and it sets a starting point for further extension of the b ion series.     

Liquid chromatography-mass spectrometry with collision-induced dissociation of conjugated metabolites of benzol[a]pyrene

Benzo[a]pyrene (BP) metabolites conjugated with glutathione, cysteine-glycine, cysteine, N-acetylcysteine, and sulfuric and glucuronic acids have been studied by microcolumn liquid chromatography-electrospray mass spectrometry with collision-induced dissociation (CID) on a hybrid double focusing magnetic sector-orthogonal time-of-flight tandem mass spectrometer equipped with a focal plane array detector. Negative-ion electrospray mass spectra of the conjugated BP metabolites showed strong [M – H]^? ions. When the array detector was used, spectra were obtained from femtomoles of sample infused at mass resolutions of 5000 (full width at half maximum). Cone voltage fragmentation spectra show [M-H]^? ions and fragment ions indicative of the BP moiety and/or the conjugating group. Linked scan CID spectra at constant B/E were found to contain structurally informative product ions from infusion of as little as 1 pmol of sample. CID spectra were also recorded by using the double focusing sectors for precursor ion selection and the orthogonal time-of-flight analyzer for product ion mass separation. The method was applied to the analysis of conjugated BP metabolites in the urine of germ-free rats given a single intraperitoneal dose of BP.     

The reaction intermediate spectrum. A new type of experiment in tandem mass spectrometry

A new scan is described which responds to ions that are intermediates in the dissociation of a mass-selected parent ion (m_p) to give a mass-selected daughter ion (m_d). The scan gives a simple mass v. abundance output for ions which satisfy this condition. It is implemented here on a BEQQ hybrid mass spectrometer using, in sequence, collision-induced dissociation occurring at high energy in the first reaction region, and low-energy collisional activation in the collision quadrupole. The experiment provides information on reaction sequences not available from single scans of other types. In the several cases examined, it is demonstrated that, among many conceivable fragmentation routes connecting a parent ion with a particular fragment ion, only a few are significant. Examination of reaction intermediate spectra also appears to be a fruitful new approach to mechanistic questions, as illustrated by consideration of the behavior of several isomeric octanones. These new spectra also have analytical value: they show good signal-to-noise ratios and allow ready distinction between isobaric and isomeric ions. A comparison of the reaction intermediate spectrum with a daughter spectrum obtained by the B/E linked-scanning technique reveals the contributions of artifact peaks which result from poor parent ion mass resolution in the latter. Reaction intermediate spectra combine information from the daughter spectra of m_p and the parent spectra of m_d and, as a specified portion of this data domain, have unique characteristics.     

A tandem mass spectrometric study of the N-oxides, quinoline N-oxide, carbadox, and olaquindox, carried out at high mass accuracy using electrospray ionization

A mass spectrometric study of three N-oxides, quinoline N-oxide, and the synthetic antibiotics carbadox and olaquindox, was carried out with a hybrid quadrupole/time-of-flight (TOF) mass spectrometer coupled with electrospray (ES) and atmospheric pressure chemical ionization (APCI) sources. The full scan mass spectra of the N-oxides obtained with ES are similar to those obtained with APCI, and the characteristic fragment ions corresponding to [M+H−O]⁺√ were observed in the full scan mass spectrum of each N-oxide examined. The protonated molecule of each N-oxide was subjected to collision-induced dissociation (CID) and accurate mass measurements were made of each fragment ion so as to determine its elemental composition. Fragment ions generated at enhanced cone voltages upstream of the first mass-resolving element were subjected to CID so as to identify the direct product ion–precursor ion relationship. Plausible structures have been proposed for most of the fragment ions observed. Elimination of OH√ radicals generated from the N→O functional group is a characteristic fragmentation pathway of the N-oxides. The expulsion of radicals and small stable molecules is accompanied by formation and subsequent contraction of heterocyclic rings.     

Characterization of the Retro-[3+2] Reaction of Protonated 2,3′-Bisindolylmethanes by Electrospray Ionization–Tandem Mass Spectrometry

Twelve 2,3′-bisindolylmethanes with various substituents were investigated using electrospray ionization quadrupole time-of-flight tandem mass spectrometry in positive ion mode. A retro-[3+2] reaction was observed in the collision-induced dissociation spectra of protonated 2,3′-bisindolylmethanes for the first time. The mechanism of retro-[3+2] reaction was concerted or stepwise. For the concerted pathway, carbon–carbon bonds of a protonated compound simultaneously cracked and the m/z 208 ion ([C₁₅H₁₀D₂N]⁺) was observed with hydrogen–deuterium exchange labeling. The stepwise pathway goes through 1,3-hydrogen migration twice and the m/z 208 ion ([C₁₅H₁₀D₂N]⁺) and m/z 207 ion ([C₁₅H₁₁DN]⁺) were detected with deuterium labeling. In the deuterium-labeled tandem mass spectrum for one compound, only the peak at m/z 208 was present at high abundance, suggesting that the concerted pathway is more likely. In addition, the substituents have no obvious trends on the ratios of the product intensity to the base intensity, further supporting the concerted pathway.     

Comparison of Triple Quadrupole and Double Focusing Mass Spectrometers to Investigate Collision-Induced Dissociation and Metastable Ions of Glycoconjugates

Glycoconjugates, such as chromophore-labeled disaccharides and permethylated glycosphingolipids (GSL) were used for comparison of triple quadrupole and double focusing mass spectrometers in analysis of product ions. A profound effect of collision energy was observed in the product ion spectra of ceramide ions (fragment ions of permethylated GSL): more product ions were observed from a double focusing mass spectrometer. Besides collision energy, the structure of the analyte had a significant effect on the formation of product ions. Despite the fact that masses of protonated molecular ions (MH⁺) of permethylated GSL are significantly larger than their ceramide fragments, the low-energy and high-energy product ion spectra of MH⁺ are, in general, similar. In a double focusing mass spectrometer of reversed geometry, more metastable ions were observed in the first field free region (FFR) than in the second FFR. The metastable ions observed in the second FFR were similar to those observed in low-energy collision-induced dissociation (CID). Although a double focusing mass spectrometer is superior to triple quadrupole instrument for detection of product ions, the poor resolution in either the selection of precursor ion or in the product ion spectra can be a serious problem in analysis of a mixture with similar masses.     

Gas-phase fragmentation reactions of protonated glycerol and its oligomers: Metastable and collision-induced dissociation reactions,associated deuterium isotope effects and the structure of [C3H5O]+, [C2H5O]+, [C2H4O]+. and [C2H3O]+ ions

The chemistry of glycerol subjected to a high-energy particle beam was explored by studying the mass spectral fragmentation characteristics of gas-phase protonated glycerol and its oligomers by using tandem mass spectrometry. Both unimolecular metastable and collision-induced dissociation reactions were studied. Collision activation of protonated glycerol results in elimiation of H₂O and CH₃OH molecules. The resulting ions undergo further fragmentations. The origin of several fragment ions was established by obtaining their product and precursor ion spectra. Corresponding data for the deuterated analogs support those results. The structures of the fragment ions of compositions [C₃H₅O]⁺, [C₂H₅O]⁺, [C₂H₄O]^+. and [C₂H₃O]⁺ derived from protonated glycerol were also identified. Proton-bound glycerol oligomers fragment principally via loss of neutral glycerol molecules. Dissociation of mixed clusters of glycerol and deuterated glycerol displays normal secondary isotope effects.     

Investigation of lacidipine,a new 1,4-dihydropyridine antihypertensive agent and three chemically related compounds by means of chemical ionization,mass spectrometry and collision-induced dissociation

Chemical ionization of two 1,4-dihydropyridines, lacidipine and its Z-isomer, and their corresponding pyridines in three different reagent gases and the collision-induced dissociation (CID) of their respective mass-selected protonated molecular ions in the collision energy range 10–200 eV were performed on a multiple quadrupole instrument. The weakness of the Breasted acid NH₄⁺ as a protonating agent is clearly manifested in one of the ammonia positive-ion chemical ionization (CI⁺) mass spectra which displays the addition ion, [M + NH₄]⁺, as the favoured reaction channel. The stereochemistry of the precursor molecules, the exothermicity of the protonation process and the threshold of certain dissociation channels as a function of the collision energy are among the arguments invoked to explain some of the observed differences between the CI⁺ mass spectra and the CID data of the different isomers investigated. In an attempt to present a more comprehensive study, some high-performance liquid chromatographic retention times and resolutions are also given.     

Reactions within and between adjacent dimethylamino groups: Collision-induced dissociation tandem mass spectrometric study of the 1,9-bis(dimethylamino)phenalenium cation

The electron impact (EI) ionization-induced fragmentation pathways of the new 1,9-bis(dimethylamino) phenalenium cation [1]⁺ were investigated. The peri-dimethylamino substituents of [1]⁺ are incorporated in a trimethine cyanine substructure and show strong steric interactions. A mechanism is proposed for the unusual elimination of CH₃N?CH₂, HN(CH₃)₂ and (CH₃)₃N from [1]⁺ and for the accompanying cyclizations to heterocyclic ions: prior to fragmentation, the intact cation [1]⁺ rearranges, by reciprocal CH₃ and H transfers, to new isomeric cations which decompose subsequently in a characteristic way. A wealth of consistent information on dissociation pathways and fragment structures is provided by collision-induced dissociation tandem mass spectra, collision-induced dissociation mass-analysed ion kinetic energy spectra and exact mass measurements of the salt cation and of its primary fragment ions. The liquid secondary ion mass spectrum of [1]⁺ is very similar to its EI mass spectrum.     

Fragmentation of protonated O,O-dimethyl O-aryl phosphorothionates in tandem mass spectral analysis

A study was carried out on the fragmentation of 12 protonated O,O-dimethyl O-aryl phosphorothionates by tandem quadrupole mass spectrometry. Some of the studied compounds are used in agriculture as pesticides. Energy-resolved and pressure-resolved experiments were performed on the [M + H]⁺ ions to investigate the dissociation behavior of the ions with various amounts of internal energy. On collisionally activated dissociation, the [M + H]⁺ ions decompose to yield the [M + H ? CH₃OH]⁺, (CH₃O)₂PS⁺ (m/z 125), and (CH₃O)₂PO⁺ (m/z 109) ions as major fragments. The ions [M + H ? CH₃OH]⁺ and (CH₃O)₂PS⁺ probably arise from the [M + H]⁺ ions of the O,O-dimethyl O-aryl phosphorothionates with the proton on the sulfur or on the oxygen of the phenoxy group. The origin of the hydroxy proton of the methanol fragment was in many cases, surprisingly, the phenyl group and not the reagent gas. This was confirmed by using deuterated isobutane, C₄D₁₀, as reagent gas in Cl. The fragment ions (CH₃O)₂PO⁺ and [ZPhS]⁺ are the results of thiono-thiolo rearrangement reaction. The precursor ion for the ion (CH₃O)₂PO⁺ arises from most compounds upon chemical ionization, whereas the precursor ion for the ion [ZPhS]⁺ arises only from a few compounds upon chemical ionization. The observed fragments imply that several sites carry the extra proton and that these sites get the proton usually upon ionization. The stability order and some characteristics of three protomers of O,O-dimethyl O-phenyl phosphorothionate were investigated by ab initio calculations at the RHF/3-21G* level of theory.     

Reactivity of gaseous sodiated ions derived from benzene dicarboxylate salts toward residual water in the collision gas

The sodium adduct of disodium salts of benzene dicarboxylic acids (m/z 233), when subjected to collision‐induced dissociation (CID), undergoes a facile loss of CO₂ to produce an ion of m/z 189, which retains all the three sodium atoms of the precursor. The CID spectrum of this unusual m/z 189 ion shows significant peaks at m/z 167, 63 and 85. The enigmatic m/z 167 ion, which appeared to represent a loss of a 22‐Da neutral fragment from the precursor ion is in fact a fragment produced by the interaction of the m/z 189 ion with traces of water present in the collision gas. The change of the m/z 167 peak to 168, when D₂O vapor was introduced to the collision gas of a Q‐ToF instrument, proved that such an intervention of water could occur even in collision cells of tandem‐in‐space mass spectrometers. The m/z 189 ion has such high affinity for water; it forms an ion/molecule complex even during the brief residence time of ions in collision cells of triple quadrupole instruments. The complex formed in this way then eliminates elements of NaOH to produce the ion observed at m/z 167. In an ion trap, the relative intensity of the m/z 167 peak increases with longer activation time even at the lowest possible collision energy setting. Similarly, the m/z 145 ion (which represents the sodium adduct of phenelenedisodium, formed by two consecutive losses of CO₂ from the m/z 233 ion of meta‐ and para‐isomers) interacts with water to produce a fragment ion at m/z 123 for the sodium adduct of phenylsodium. Other uncommon ions that originate also from water/ion interactions are observed at m/z 85 and 63 for [Na₃O]⁺ and [Na₂OH]⁺, respectively. Tandem mass spectrometric experiments conducted with appropriately deuterium‐labeled compounds confirmed that the proton required for the formation of the [Na₂OH]⁺ ion originates from traces of water present in the collision gas and not from the ring protons of the aromatic moiety. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of a new fragment ion type in the collision-induced dissociation spectra of peptides: Formation of a2-16 ions

The identification of an ion observed in the high-energy collision-induced dissociation spectra of several model peptides is reported. The ion, observed at m/z 99 for the peptide pentaalanine (Ala₅) and designated a₂-16, is shown to have an elemental formula of C₅H₉NO by high-resolution peak matching. The precursor ion spectrum of the a₂-16 ion and product ion spectra of the a₂ and the a₂+ 1 ions for Ala₅ suggest that this ion is formed by the loss of 17 u (presumably NH₃) from the a₂+1 ion and, to a lesser extent, by loss of 28 u (presumably CO) from the b₂-16 ion. On the basis of the data presented and other experimental evidence, a structure and mechanism for the formation of the a₂-16 ion is proposed.     

Negative ion tandem mass spectrometry of leukotriene E4, and LTE4, metabolites: Identification of LTE4, in human urine

The sulfidopeptide leukotrienes, leukotriene E₄, (LTE₄,) and its N-acetyl derivative and several ω? and β-oxidized metabolites of LTE₄, have been analyzed by tandem mass spectrometry. [M?H]^? ions were produced by continuous flow fast atom bombardment, and collision-induced dissociation of these ions was studied by using a triple quadrupole instrument. The product ion spectra obtained were characteristic of the structure of LTE₄, and mechanisms of ion formation were investigated by using deuterated compounds. β-Elimination of the peptide portion of LTE₄, by loss of CO₂, and ethylene amine leaves the C-l carboxyl group ionized in the most abundant fragment ion for LTE₄, and all metabolites. Tandem mass spectrometry of fast atom bombardment-generated anions from ω? and β-oxidized metabolites of LTE₄, produced similar ions with only a minor influence of the third carboxyl group at the omega terminus evident. Tandem mass spectrometry was used to identify unequivocally the presence of unmodified LTE₄, in a high performance liquid chromatography-purified fraction of urine from a normal healthy volunteer after infusion with LTE₄.