Multiplexed MS/MS in a quadrupole ion trap mass spectrometer

A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods.     

Thermally assisted infrared multiphoton photodissociation in a quadrupole ion trap

Thermally assisted infrared multiphoton photodissociation (TA-IRMPD) provides an effective means to dissociate ions in the quadrupole ion trap mass spectrometer (QITMS) without detrimentally affecting the performance of the instrument. IRMPD can offer advantages over collision-induced dissociation (CID). However, collisions with the QITMS bath gas at the standard pressure and ambient temperature cause IR-irradiated ions to lose energy faster than photons can be absorbed to induce dissociation. The low pressure required for IRMPD (< or = 10(-5) Torr) is not that required for optimal performance of the QITMS (10(-3) Torr), and sensitivity and resolution suffer. TA-IRMPD is performed with the bath gas at an elevated temperature. The higher temperature of the bath gas results in less energy lost in collisions of the IR-excited ions with the bath gas. Thermal assistance allows IRMPD to be used at or near optimal pressures, which results in an approximately 1 order of magnitude increase in signal intensity. Unlike CID, IRMPD allows small product ions, those less than about one-third the m/z of the parent ion, to be observed. IRMPD should also be more easily paired with fluctuating ion sources, as the corresponding fluctuations in resonant frequencies do not affect IRMPD. Finally, while IR irradiation nonselectively causes dissociation of all ions, TA-IRMPD can be made selective by using axial expansion to move ions away from the path of the laser beam.     

Dynamic collision-induced dissociation of peptides in a quadrupole ion trap mass spectrometer

The fragmentation of natural peptides using dynamic collision-induced dissociation (DCID), a novel fragmentation method for quadrupole ion traps, is demonstrated. Using leucine enkephalin as a diagnostic molecule, the fragmentation efficiencies and energetics of DCID are compared with other methods of collisional activation in ion traps such as conventional on-resonance excitation and high-amplitude short-time excitation (HASTE). A typical fragmentation efficiency of approximately 20% is achieved for DCID, which is significantly lower than conventional CID (maximum near 80%). Tandem mass spectra of two other peptides, substance P and oxidized insulin alpha-chain, demonstrate that product ion spectra for DCID are comparable to conventional or HASTE CID. Because DCID achieves fragmentation during the standard mass acquisition scan, no extra time is necessary for on-resonance excitation or product ion collection, so analysis times are reduced by a minimum of 10-15% depending on the scanning conditions. DCID therefore offers more tandem mass spectra per second than conventional methods of collisional activation, which could be highly advantageous for bottom-up proteomics separations.     

Peptide rearrangement during quadrupole ion trap fragmentation: added complexity to MS/MS spectra

The emergence of proteomics has placed great interest in the understanding of the mechanisms of MS/MS fragmentation of peptides under low-energy collision-induced dissociation. In this work, we describe the presence of anomalous fragments, which correspond to neutral loss elimination of internal amino acids from ions of the b series in quadrupole ion trap MS/MS spectra from naturally occurring peptides. Internal amino acid elimination occurred preferentially with aliphatic amino acids. The phenomenon was more apparent when doubly charged precursors were fragmented and was inhibited when peptides were N-acetylated at the N-terminus. Fragmentation of isomeric peptides where some internal amino acids were relocated in N-terminal position produced MSn spectra indistinguishable from those of the original peptides, indicating that some b ions underwent a structural rearrangement process. Formation of anomalous fragments required a minimum activation time. Our data are consistent with a nucleophile attack of the N-terminal nitrogen over the electrophilic carbonyl carbon at one peptide bond, forming a cyclic b ion intermediate that, by reopening at preferential sites, exposes internal amino acids to the C-terminal side.     

193 nm ultraviolet photodissociation of imidazolinylated Lys-N peptides for de novo sequencing

The goal of many MS/MS de novo sequencing strategies is to generate a single product ion series that can be used to determine the precursor ion sequence. Most methods fall short of achieving such simplified spectra, and the presence of additional ion series impede peptide identification. The present study aims to solve the problem of confounding ion series by enhancing the formation of "golden" sets of a, b, and c ions for sequencing. Taking advantage of the characteristic mass differences between the golden ions allows N-terminal fragments to be readily identified while other ion series are excluded. By combining the use of Lys-N, an alternate protease, to produce peptides with lysine residues at each N-terminus with subsequent imidazolinylation of the ε-amino group of each lysine, peptides with highly basic sites localized at each N-terminus are generated. Subsequent MS/MS analysis by using 193 nm ultraviolet photodissociation (UVPD) results in enhanced formation of the diagnostic golden pairs and golden triplets that are ideal for de novo sequencing.     

Ultraviolet photodissociation at 355 nm of fluorescently labeled oligosaccharides

Ultraviolet photodissociation (UVPD) produces complementary fragmentation to collision-induced dissociation (CID) when implemented for activation of fluorescently labeled oligosaccharide and glycan ions. Reductive amination of oligosaccharides with fluorophore reagents results in efficient photon absorption at 355 nm, producing fragment ions from the nonreducing end that do not contain the appended fluorophore. In contrast to the fragment ions observed upon UVPD (A- and C-type ions), CID produces mainly reducing end fragments retaining the fluorophore (Y-type ions). UVPD affords better isomeric differentiation of both the lacto-N-fucopentaoses series and the lacto-N-difucohexaoses series, but in general, the combination of UVPD and CID offers the most diagnostic elucidation of complex branched oligosaccharides. Four fluorophores yielded similar MS/MS results; however, 6-aminoquinoline (6-AQ), 2-amino-9(10 H)-acridone (AMAC) and 7-aminomethylcoumarin (AMC) afforded more efficient photon absorption and subsequent dissociation than 2-aminobenzamide (2-AB). UVPD also was useful for characterization of glycans released from ribonuclease B and derivatized with 6-AQ. Lastly, electron photodetachment dissociation of oligosaccharides derivatized with 7-amino-1,3-naphthalenedisulfonic acid (AGA) yielded unique cross-ring cleavages similar to those obtained by electron detachment dissociation.     

Structural characterization of neutral glycosphingolipids by thin-layer chromatography coupled to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight MS/MS

Rapid and convenient structural analysis of neutral glycosphingolipids (GSLs) was achieved by direct coupling of thin-layer chromatography (TLC) to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) MS/MS. Positions of unstained GSL spots on developed TLC plates were determined by comparison to orcinol-stained references. A matrix solution of 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/water (1:1 v/v) was then added directly to the unstained GSL spots, and the GSLs were directly analyzed by MALDI-QIT-TOF MS. The acetonitrile/water DHB solution proved to be suitable for MS/MS structural analysis with high sensitivity. MS/MS and MS/MS/MS of GSLs yielded simple and informative spectra that revealed the ceramide and long-chain base structures, as well as the sugar sequences. Hydroxy fatty acids in ceramide provided characteristic MS/MS fragment ions. GSLs were stained with primuline, a nondestructive dye, after TLC development, and successfully analyzed by MALDI-QIT-TOF MS/MS with high sensitivity. Immunostaining of GSLs after TLC development is a powerful method for characterizing antibody-specific sugars, but not ceramides. By coupling TLC-immunostaining of GSLs to MALDI-QIT-TOF MS/MS, we were able to identify both the sugar and the ceramide structures. The detection limits of asialo GM1 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1-1'Cer) were 25 and 50 pmol in primuline staining and immunostaining, respectively.     

Oligonucleotide mixture analysis via electrospray and ion/ion reactions in a quadrupole ion trap

Electrospray ionization combined with ion/ion reactions in a quadrupole ion trap can be used for the direct analysis of oligonucleotide mixtures. Elements to the success of this approach include factors related to ionization, ion/ion reactions, and mass analysis. This paper deals with issues regarding the ion polarity combination, viz., positive oligonucleotides/negative charge-transfer agent versus negative oligonucleotides/positive charge-transfer agent. Anions derived from perfluorocarbons appear to be directly applicable to mixtures of positive ions derived from electrospray of oligonucleotides, in direct analogy with positive protein ions. Conditions for forming positive oligonucleotide ions devoid of adducts were more difficult to establish than for forming relatively clean negative oligonucleotide ions. A new approach for manipulating negative ion charge states in the ion trap is described and is based on use of the electric field of the positive charge-transfer agent for storage of high-mass negative ions formed during the ion/ion reaction period. Oxygen cations are shown to be acceptable for charge-state manipulation of mixed-base oligomers but induce fragmentation in polyadenylate homopolymers. Protonated isobutylene (C4H9+), on the other hand, is shown to induce significantly less fragmentation of polyadenylate homopolymers.     

Differentiation of phosphorylated and unphosphorylated peptides by high-performance liquid chromatography-electrospray ionization-infrared multiphoton dissociation in a quadrupole ion trap

Infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap coupled to high-performance liquid chromatography allows the selective dissociation of phosphorylated peptides in mixtures following chromatographic separation. This method is shown to be effective for differentiation of phosphorylated peptides from unphosphorylated ones; only the abundances of the phosphorylated species are appreciably decreased following exposure to 125 ms of 10.6-microm radiation. This LC-IRMPD-MS strategy is demonstrated for a mock mixture of peptides and a tryptic digest of alphaS1-casein. The ability of this technique to differentiate peptides based on phosphorylation state is unaffected by whether the peptides are protonated or sodium-cationized.     

Calibration of ion effective temperatures achieved by resonant activation in a quadrupole ion trap

The present paper describes a calibration of the ion effective temperatures as a function of the resonant activation amplitude in a quadrupole ion trap mass spectrometer. MS/MS experiments are performed on leucine enkephalin (M + H)+, bradykinin (M + H)+, (M + 2H)2+, and (M + 3H)3+, and ubiquitin (M + 11H)11+. For each amplitude, the effective temperature is calculated as the temperature that would give the same dissociation rate constant as the one observed and is calculated using published Arrhenius parameters. The effective temperature is found to be linearly dependent on the activation amplitude on the range investigated. The dependence of the slope and of the intercept of the T(eff) = f (amplitude) functions on the parent ion m/z is examined and an equation is derived to calibrate the ion effective temperature between 365 and 600 K. Below 365 K, a deviation from linearity is expected. Above 600 K, the validity of the equation will depend on whether the rapid energy exchange limit is still reached. Calculating backward, the Arrhenius parameters from the measured dissociation rates using this calibration show excellent agreement with the published values. The calibration can therefore be used to determine Arrhenius activation parameters from dissociation kinetics under resonant activation in quadrupole ion trap mass spectrometers.     

Fourier transform detection in a cylindrical quadrupole ion trap

Broad-band nondestructive ion detection based on induced image current measurement is performed in a quadrupole ion trap having cylindrical geometry. Spectra of krypton and acetophenone are shown to demonstrate the first use of nondestructive detection with a cylindrical ion trap.     

Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*-

Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.     

193 nm ultraviolet photodissociation of deprotonated sialylated oligosaccharides

The fragmentation patterns of deprotonated sialylated oligosaccharides and glycans from fetuin obtained upon collisionally induced dissociation (CID) and 193 nm ultraviolet photodissociation (UVPD) in a linear ion trap are presented. UVPD produced a more extensive series of cross-ring cleavage ions, such as A- and X-type ions, and dual-cleavage internal ions, including A/Y and X/B fragment ions. In addition, UVPD generated unique fragment ions which arise from site-specific cleavage of the triol substituent of the sialic acid residue. In contrast, CID produced more conventional glycosidic cleavages and relatively few A-type ions. UVPD of doubly deprotonated sialylated oligosaccharides produced mostly singly deprotonated fragment ions, whereas the product ions in the CID spectra were overwhelmingly doubly charged ions, an outcome attributed to the more extensive cleavages of sialic acid residues upon UVPD and products from electron photodetachment. The larger array of product ions, including those arising from extensive cross-ring cleavages and dual-cleavage ions, generated by 193 nm UVPD relative to CID gives greater confidence for identification of glycans. Several key site-specific cleavages by UVPD, such as ones involving the sialic acid moieties, provide evidence of glycan composition.     

Use of a designed peptide array to infer dissociation trends for nontryptic peptides in quadrupole ion trap and quadrupole time-of-flight mass spectrometry

Observed peptide gas-phase fragmentation patterns are a complex function of many variables. To systematically probe this phenomenon, an array of 40 peptides was synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant and randomize or balance others (peptide amino acid distribution and position). A high-quality tandem mass spectrometry (MS/MS) data set was acquired for each peptide for all observed charge states on multiple MS instruments, quadrupole-time-of-flight and quadrupole ion trap. The data were analyzed as a function of total charge state and number of mobile protons. Previously known dissociation trends were observed, validating our approach. In addition, the general influence of basic amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic peptides, the amino acids H, K, and R were positionally distributed. Interestingly, our results suggest that cleavage at all basic amino acids is suppressed when a mobile proton is available. Cleavage at H becomes favored only under conditions where a partially mobile proton is present, a caveat to the previously reported trend of enhanced cleavage at H. Finally, all acquired data were used as a benchmark to determine how well these sequences would have been identified in a database search using a common algorithm, Mascot.     

Infrared multiphoton dissociation of duplex DNA/drug complexes in a quadrupole ion trap

Noncovalent duplex DNA/drug complexes formed between one of three 14-base pair non-self-complementary duplexes with variable GC content and one of eight different DNA-interactive drugs are characterized by infrared multiphoton dissociation (IRMPD), and the resulting spectra are compared to conventional collisionally activated dissociation (CAD) mass spectra in a quadrupole ion trap mass spectrometer. IRMPD yielded comparable information to previously reported CAD results in which strand separation pathways dominate for complexes containing the more AT-rich sequences and/or minor groove binding drugs, whereas drug ejection pathways are prominent for complexes containing intercalating drugs and/or duplexes with higher GC base content. The large photoabsorptive cross section of the phosphate backbone at 10.6 mum promotes highly efficient dissociation within short irradiation times (<2 ms at 50 W) or using lower laser powers and longer irradiation times (<15 W at 15 ms), activation times on par with or shorter than standard CAD experiments. This large photoabsorptivity leads to a controllable ion activation method which can be used to produce qualitatively similar spectra to CAD while minimizing uninformative base loss dissociation pathways or instead be tuned to yield a high degree of secondary fragmentation. Additionally, the low-mass cutoff associated with conventional CAD plays no role in IRMPD, resulting in richer MS/MS information in the low m/z region. IRMPD is also used for multiadduct dissociation in order to increase MS/MS sensitivity, and a two-stage IRMPD/IRMPD method is demonstrated as a means to give specific DNA sequence information that would be useful when screening drug binding by mixtures of duplexes.     

Infrared multiphoton dissociation for enhanced de novo sequence interpretation of N-terminal sulfonated peptides in a quadrupole ion trap

Infrared multiphoton dissociation (IRMPD) of N-terminal sulfonated peptides improves de novo sequencing capabilities in a quadrupole ion trap mass spectrometer. Not only does IRMPD promote highly efficient dissociation of the N-terminal sulfonated peptides but also the entire series of y ions down to the y(1) fragment may be detected due to alleviation of the low-mass cutoff problem associated with conventional collisional activated dissociation (CAD) methods in a quadrupole ion trap. Commercial de novo sequencing software was applied for the interpretation of CAD and IRMPD MS/MS spectra collected for seven unmodified peptides and the corresponding N-terminal sulfonated species. In most cases, the additional information obtained by N-terminal sulfonation in combination with IRMPD provided significant improvements in sequence identification. The software sequence tag results were combined with a commercial database searching algorithm to interpret sequence information of a tryptic digest on alpha-casein s1. Energy-variable CAD studies confirmed a 30-40% reduction in the critical energies of the N-terminal sulfonated peptides relative to unmodified peptides. This reduction in dissociation energy facilitates IRMPD in a quadrupole ion trap.     

Collision-activated infrared multiphoton dissociation in a quadrupole ion trap mass spectrometer

We propose and demonstrate a new method for multiple-stage mass spectrometry (MSn), collision-activated infrared multiphoton dissociation (CA-IRMPD), which is very effective for the quadrupole ion trap mass spectrometer (QITMS). CA-IRMPD uses a combination of focused laser irradiation (beam radius, approximately 0.4 mm) and collisional activation by a supplemental AC voltage between endcap electrodes. This combination enables IRMPD, which has conventionaLly been ineffective above 10(-4) Torr, to be used under a standard bath gas pressure of 2-8 mTorr. CA-IRMPD can produce richer spectra of product ions than CID or IRMPD while maintaining high sensitivity and mass resolution; thus, it will contribute to an accurate determination of peptide sequences.     

Thermally assisted collision-induced dissociation in a quadrupole ion trap mass spectrometer

Thermally assisted collision-induced dissociation (TA-CID) provides increased dissociation in comparison with CID performed at ambient temperature in a quadrupole ion trap mass spectrometer. Heating the bath/collision gas during CID increases the initial internal energy of the ions and reduces the collisional cooling rate. Thus, using the same CID parameters, the parent ion can be activated to higher levels of internal energy, increasing the efficiency of dissociation and the number of dissociation pathways. The increase in the number of dissociation pathways can provide additional structural information. A consequence of the increase in initial internal energy is the ability to use less power to effect collisional activation. This allows lower q(z) values to be used and, thus, a greater mass range of product ions to be observed. TA-CID alleviates the problems associated with traditional CID and results in more available information than traditional CID.     

Dynamic pressure measurements during pulsed gas injection in a quadrupole ion trap

A quadrupole ion trap has been modified to perform dynamic pressure measurements during pulsed introduction of gases. A continuous electron beam is directed through the ion trap where the gas is ionized via electron impact. Ion and electron currents are monitored on the ring and end-cap electrodes, respectively. Dynamic pressure measurements in a region not accessible to a standard gauge are performed using a static quadrupole field. Characteristic current-voltage curves of the ion-trap gauge are presented and optimum operating conditions of the electron-ion optical system are identified in steady state conditions. The sensitivity of the ion-trap gauge is calibrated at these optimum conditions. In the pulsed gas mode ion and electron signals are measured simultaneously on a fast oscilloscope. The time constant of the circuit for the dynamic measurements is ∼129 μs and pressure variations of ∼10⁻⁴-1 Pa occur within 60-130 ms. The exponential decay of the ion signals is used to calculate pumping speeds for helium and argon gases. The distinctive advantages of pulsed gas injections over the use of static pressures in quadrupole ion-trap mass spectrometry are emphasized.     

Electron photodetachment dissociation of DNA polyanions in a quadrupole ion trap mass spectrometer

We hereby explore the effects of irradiating DNA polyanions stored in a quadrupole ion trap mass spectrometer with an optical parametric oscillator laser between 250 and 285 nm. We studied DNA 6-20-mer single strands and 12-base pair double strands. In all cases, laser irradiation causes electron detachment from the multiply charged DNA anions. Electron photodetachment efficiency directly depends on the number of guanines in the strand, and maximum efficiency is observed between 260 and 275 nm. Subsequent collision-induced dissociation (CID) of the radical anions produced by electron photodetachment results in extensive fragmentation. In addition to neutral losses, a large number of fragments from the w, d, a*, and z* ion series are obtained, contrasting with the w and (a-base) ion series observed in regular CID. The major advantage of this technique, coined electron photodetachment dissociation (EPD) is the absence of internal fragments, combined with good sequence coverage. EPD is therefore a highly promising approach for de novo sequencing of oligonucleotides. EPD of nucleic acids is also expected to give specific radical-induced strand cleavages, with conservation of other fragile bonds, including noncovalent bonds. In effect, preliminary results on a DNA hairpin and on double strands suggest that EPD could also be used to probe intra- and intermolecular interactions in nucleic acids.