Phase correction for collision model analysis and enhanced resolving power of fourier transform ion cyclotron resonance mass spectra

Phase correction of FT-ICR data yields an absorption spectrum that offers a gain by up to a factor of 2 in mass resolving power (at half-maximum peak height), compared to conventional magnitude-mode display. That improvement is equivalent to doubling the applied magnetic field strength, without loss in signal-to-noise (S/N) ratio, provided that the time-domain data are padded with an equal number of zeroes before FFT. Our simple, visual, user-interactive algorithm quickly corrects for zero-order and first-order variation of phase with frequency. We find that the theoretical mass resolving power enhancement for pressure-limited absorption-mode over magnitude-mode line shape depends on the collision mechanism: factor of 1.40 for hard sphere vs 3(1/2) for Langevin (ion: induced dipole). Thus, the experimental enhancement in mass resolving power (factor of 1.43 +/- 0.09) for isotopically resolved peaks in the FT-ICR mass spectra of electrosprayed bovine carbonic anhydrase (approximately 29 kDa) directly supports the hard-sphere collision model. Optimal implementation of phasing requires the following: (a) a delay between excitation and detection of less than half of one sampling interval to avoid baseline "roll" and Gibb's oscillations; (b) accurate analog-to-digital conversion; (c) a sufficiently long acquisition period to yield several data points per absorption-mode peak width at half-maximum peak height; and (d) avoidance of FT-ICR apodization functions (e.g., Hamming and Hanning) that suppress the initial time-domain data. Pulsed single-frequency excitation (duration much less than the reciprocal of the Nyquist bandwidth) can eliminate higher than first-order variation of phase with frequency. Phased FT-ICR spectra should prove especially desirable for analysis of complex mixtures, for resolving isotopic distributions in electrosprayed multiply charged macromolecules and for characterizing ion collisions (and thus ion size and shape).     

Procedures for tandem mass spectrometry on an ion trap storage/reflectron time-of-flight mass spectrometer

In this work, we demonstrate tandem mass spectrometry on an ion trap storage-reflectron time-of-flight mass spectrometer (IT/reTOFMS). Ion isolation and activation were achieved by resonant excitation using multi- and single-frequency waveforms generated from an analog circuit. Product-ion spectra of small polypeptides are obtained, which are comparable in fragmentation to those acquired on sector or hybrid mass spectrometers. Several important parameters governing the tandem mass spectrometry process, including the activation tickle voltage, type of collision gas, activation period and cooling period after the fragmentation were optimized using leucine-enkephalin as a model. Although the limited energy deposition from collisional activation in our experiments does not allow efficient fragmentation of large singly charged polypeptides with m/z higher than 1000, the problem may be partially solved by taking advantage of fragmenting the multiply charged ions produced from the electrospray ionization source as demonstrated for a synthetic polypeptide of molecular weight 2782. Compared to the singly charged form, the reduced m/z of multiply charged forms experience a greater trapping force as described by the pseudopotential well-depth model. Increased pseudopotential well-depths for multiply charged species permit the use of greater fragmentation energy at lower RF potentials. These conditions facilitate the fragmentation of large polypeptides, yet are suitable for trapping singly charged fragments. These experiments indicate that the high efficiency associated with ion dissociation and fragment-ion collection in the trap and the storage capability for detection of ions using the non-scanning mode of the IT/ reTOF analyzer may provide an alternative means for acquiring sequence-specific information of polypeptides at low picomol levels of sensitivity.     

C-terminal peptide sequencing via multistage mass spectrometry

Results are presented showing the ability to obtain C-terminal sequence information from peptides by multiple stages of mass spectrometry. Under typical low-energy collision-induced dissociation conditions of quadrupole ion trap and ion cyclotron resonance mass spectrometers, lithium- and sodium-cationized peptides dissociate predominantly by reaction at the C-terminal peptide bond or an adjacent bond. For the majority of cases studied, the dominant reaction is a rearrangement process that results in the loss of the C-terminal residue and formation of a product ion that is one amino acid shorter than the original peptide ion. Using the multistage MS/MS capabilities of quadrupole ion trap and ion cyclotron resonance mass spectrometers, a subsequent stage of MS/MS can be performed to determine the identity of the new C-terminal residue. Up to eight stage of MS/MS have been performed with both quadrupole ion trap and ion cyclotron resonance mass spectrometers. In general, the same dissociation pathways are observed with both instruments, although occasionally there are significant differences in the branching ratios of competing pathways.     

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.     

Quantitative subfemtomole analysis of alpha-tocopherol and deuterated isotopomers in plasma using tabletop GC/MS/MS

A rapid, high-selectivity method with subfemtomole sensitivity is reported for quantification of alpha-tocopherol in plasma-based gas chromatography/tandem mass spectrometry (GC/MS/MS) using a tabletop quadrupole ion trap mass spectrometer. Sample workup is rapid, consisting of protein precipitation followed by liquid/liquid extraction and O-trimethylsilyl derivatization of alpha-tocopherol (alpha-T-TMS) and an internal standard, 2,2,5,7,8-pentamethyl-6-chromanol (PC-TMS). Rudimentary chromatography was carried out using an 8-m DB-5 capillary column resulting in an analyte retention time of 7.2 min. No interferences from the plasma matrix were observed. The assay has a detection limit of 178 amol (89.6 fg) and a lower limit of quantification of 700 amol (350 fg) of derivatized alpha-tocopherol in diluted plasma; < 30 pL of plasma is estimated to yield sufficient alpha-tocopherol for quantitative analysis at typical concentrations found in humans. A calibration curve constructed from National Institute of Standards and Technology serum standards was linear in the working range of 1.9-1073 ng/mL (0.95-0.54 ng). Within- and between-day precision averaged 5.8% and did not exceed 11.3% for three concentrations of quality control (QC) solutions. The overall accuracy for the QC samples was within 7.2%. Storage studies showed that, alpha-T-TMS and PC-TMS are stable under conditions that might be encountered during analyses. In a test study, plasma kinetic curves for alpha-tocopherol-d6 and alpha-tocopherol-d3 were obtained for a catheterized pregnant ewe and her fetus who were simultaneously given a bolus injection of alpha-tocopherol-d6, to the ewe and alpha-tocopherol-d3 to the fetus. These data show that a tabletop GC ion trap can determine alpha-T-TMS and its isotopomers quantitatively at high selectivity in a complex matrix.     

Trace quantitation of the novel cholinesterase inhibitor in human plasma by capillary gas chromatography/ion trap mass spectrometry

This paper demonstrates the utility of an ion trap mass spectrometer as a detector for trace quantitative determinations of pharmaceuticals in human plasma by capillary gas chromatography/mass spectrometry. A novel acetylcholinesterase inhibitor (CI-1002) was selected as an illustrative example for the technique. When coupled with a selective solid-phase extraction, this approach was capable of quantifying as little as 34 pg (0.50 ng ml-1, RSD = 12.7%) of compound on the column, and the inter-run precision was typically 3-4% RSD over a 0.5-25 ng ml-1 linear range. The advantages and requirements of the technique, in addition to the prospects for improvements in the detection limit, are discussed.     

De novo peptide sequencing in an ion trap mass spectrometer with 18O labeling

De novo peptide sequencing in an ion trap mass spectrometer coupled on-line with a capillary HPLC using 18O labeling provides a viable alternative to the method using the combination of nanospray, 18O labeling and a quadrupole/time-of-flight mass spectrometer. Seven to sixteen amino acid residues can be sequenced from the liquid chromatography/randem mass spectrometry (LC/MS/MS) spectra. This approach combines the benefit of capillary LC and the high sensitivity of the ion trap operated in the MS/MS mode. The wide availability of the LCQ mass spectrometer makes this approach readily adaptable to the biological mass spectrometry community.     

Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with on-line electrospray time-of-flight mass spectrometry

Reversed-phase liquid chromatography (LC) using a nonporous silica support has been combined with electrospray (ES) time-of-flight (TOF) mass spectrometry (MS) for the fast separation and mass detection of peptides. Using this LC method, the resolution of a peptide mixture can be completed is less than 35 s. The resulting chromatographic peak widths are less than 1 s wide. Because of the unique nature of a TOF mass analyzer, complete mass spectra can be acquired at a rate which is sufficient to sample these narrow peaks. When compared with conventional LC, the same separation takes nearly 20 min to complete, and the signal-to-noise ratio observed in the total ion chromatogram is dramatically lower due to the influence of increased background noise in the mass spectra. The limit of detection for a low molecular weight peptide, Val-Pro-Leu, was found to be 6 pmol with the total ion chromatogram and 500 fmol with the reconstructed ion chromatogram. A peptide map of horse heart myoglobin, completed in 3.5 min, is shown as an example of the results which can be obtained from combining this fast LC method with fast ES/TOF/MS detection capability.     

Ion/ion proton transfer reactions for protein mixture analysis

Ion/ion proton transfer reactions are shown to be an effective means to facilitate the resolution of ions in electrospray mass spectrometry that differ in mass and charge but are similar in mass-to-charge ratio. Examples are shown in which a minor contaminant protein in a ribonuclease B solution is clearly apparent after ion/ion proton transfer but not in the conventional electrospray mass spectrum. A further example involving a mixture of bovine serum albumin and bovine transferrin also showed the identification of previously unnoticed "contaminant" polymer. The latter mixture also illustrated important issues in the use of the quadrupole ion trap as a reaction vessel and mass analyzer for high mass-to-charge ratio ions. The results suggest that the use of ion trap operating parameters specifically tailored for storage, ejection, detection, and mass-to-charge analysis of high mass-to-charge ratio ions can have attractive analytical figures of merit for determining mixtures of relatively high-mass proteins and, by extension, other types of high-mass biopolymers.     

The structure of free radical metabolites detected by EPR spin trapping and mass spectroscopy from halocarbons in rat liver microsomes

Electron impact (EI) tandem mass spectrometry (MS/MS) combined with EPR spin trapping was used to detect and identify the free radical metabolites of various halocarbons in rat liver microsomal dispersions. EPR spectra of the spin adducts of radical metabolites derived from fluorine-containing halocarbons display fluorine hyperfine splitting, which can be used as proof for the identification of this kind of halocarbon-derived free radical spin adduct. For halocarbons without fluorine atoms, MS/MS was found to be a very useful and simple method for the detection and identification of the structures of halocarbon-derived spin adducts from radical metabolites. The molecular ions from spin adducts of these halocarbon-derived free radical intermediates were observed for the first time by scanning the precursor ion spectrum of m/z 57. These assignments were further confirmed by the use of perdeuterated tert-butyl PBN which provides the precursor ion spectrum of m/z 66.     

Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry

Microfabrication technology offers the opportunity to construct microfluidic modules which are designed to perform specific, dedicated functions. Here we report the construction of a microfabricated device for the generation and delivery by electroosmotic pumping of solvent gradients at nanoliter per minute flow rates. The device consists of three solvent reservoirs and channels which were etched in glass. Solvent gradients and solvent flows were generated by computer controlled differential electroosmotic pumping of aqueous and organic phase, respectively, from the solvent reservoirs. The device was integrated into an analytical system consisting of the solvent gradient delivery module, a reverse phase microcolumn and an electrospray ionization ion trap mass spectrometer (MS). The system was used for the analysis at high sensitivity of peptides and peptide mixtures generated by proteolytic digestion of proteins. We have measured an absolute limit of detection as low as 1 fmol and a concentration limit of detection at the 100 amol/microL level. The system was also successfully used for the identification of proteins separated by 1D and 2D gel electrophoresis. This was achieved by gradient frontal analysis of the peptide mixture generated by proteolysis of the respective proteins, and the automated generation and interpretation of collision-induced dissociation spectra.     

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.     

电感耦合等离子体串联质谱法测定 高纯氧化镁粉中金属杂质元素

基于电感耦合等离子体串联质谱(ICP-MS/MS)法建立了准确可靠分析高纯氧化镁粉中金属杂质元素的新方法。ICP-MS/MS通过启用新的质量过滤装置,在氧化镁基质的金属杂质元素测定过程中,能有效减少多原子干扰。采用He为碰撞气,O₂以及NH₃/He混合气为反应气,对比了在单四极杆(SQ)模式和串联四极杆(MS/MS)模式下消除干扰的效果。采用He碰撞模式无法消除一些特殊的质谱干扰,特别是双电荷离子干扰;然而,将分析物转移为氧化物离子或团簇离子,能实现待测元素的无干扰分析,并能获得极低的检出限,通过加标回收实验评估了方法的准确性。结果显示,方法的检出限为0.46~65.9ng/L。各元素的线性相关系数(R²)均不小于0.9998,真实样品的加标回收率为93%~108%,相对标准偏差为1.6%~4.4%。方法完全能用于高纯氧化镁粉中金属杂质元素的实时监控。     

Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications--further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA).     

A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry

Nanoelectrospray mass spectrometry, the infusion at low flow rates of unseparated peptide mixtures representing protein proteolytic digests into an electrospray ionization mass spectrometer (MS), has been shown to be a suitable method for the analysis of small amounts of proteins. However, the current technique is time consuming, tedious, and difficult to automate. We used microfabrication technologies to construct a device for the sequential infusion of different peptide samples into an electrospray ionization MS without the need for sample manipulation. In this device, etched sample and buffer reservoirs are connected via etched channels to microelectrospray ion source. Peptide samples, typically unseparated tryptic digests of proteins, are applied to different reservoirs. A flow of liquid originating from a specific reservoir is generated and selectively directed toward the microsprayer and the MS by electroosmotic pumping. The analyte proteins are identified by searching sequence databases with the information contained in the collision-induced spectra of selected peptides. With this system, we have achieved a limit of detection in the low femtomoles per microliter range for peptide standards. We also show that samples deposited in different reservoirs can be sequentially mobilized without cross-contamination and that proteins can be conclusively identified at the low femtomoles per microliter level. The successful coupling online of microfabricated devices to an electrospray ionization MS represents an essential step toward the construction of automated, high-throughput, and high-sensitivity analytical systems.     

Diffusion-weighted MR imaging of bone marrow: differentiation of benign versus pathologic compression fractures

A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection. The HPLC-MS approach in the single ion monitoring (SIM) mode and the HPLC-MS/MS assay in the multiple reaction monitoring (MRM) mode have been compared, using isotopically labeled [M+4] 8-oxodGuo as the internal standard. The limit of detection of 8-oxodGuo was found to be around 5 pmol and 20 fmol for the HPLC-MS and HPLC-MS/MS methods, respectively. The HPLC-MS/MS assay is sensitive enough to allow the determination of the level of 8-oxodGuo in cellular liver DNA and in urine samples.     

A MALDI probe for mass spectrometers

A new MALDI probe has been designed that uses transmission geometry. This geometry allows the probe to be fashioned after typical EI/CI solid probes which enables it to be introduced into spatially constrained ion source regions such as encountered in quadrupole ion trap mass spectrometers. In the probe design demonstrated here, light from a fiber optic irradiates the backside of a sample through a small piece of quartz on which the sample has been directly deposited. The performance characteristics exhibited by utilizing this probe for MALDI on a quadrupole ion trap mass spectrometer are similar to those which can be obtained through the traditional methods of implementing MALDI. Spectra have been obtained from 50 fmol of total loading of bombesin, MS/MS has been performed on 5 pmol of des-Arg9-bradykinin, and the analyte ion signal is shown to last for over 2500 laser shots for 2 pmol of bombesin. Optical micrographs showing the crystal distribution of a sample containing 2 pmol of bombesin have been obtained as a function of the number of laser shots for a single sample loading. Although this probe was designed for use with the quadrupole ion trap, it can be adapted for use with all types of mass spectrometers. Thus, with only one laser, one fiber optic, and this probe, MALDI can be performed on multiple instruments in a lab.     

Microfabricated device coupled with an electrospray ionization quadrupole time-of-flight mass spectrometer: protein identifications based on enhanced-resolution mass spectrometry and tandem mass spectrometry data

We describe the coupling of a microfabricated fluidic device to an electrospray ionization (ESI) quadrupole time-of-flight mass spectrometer (QqTOFMS) for the identification of protein samples. The microfabricated devices consisted of three reservoirs connected via channels to a main capillary, which in turn was linked via a microspray interface to the QqTOFMS. Here we present preliminary results obtained using this system. Standardized solutions of myoglobin tryptic digest were analyzed indicating a limit of detection at the low to sub fmol/microL. The combination of the microfabricated device for rapid sample delivery and the rapid acquisition capability, enhanced resolution and mass accuracy of the QqTOF offers unique possibilities for the rapid identification of proteins by database searching. This platform can generate MS data suitable for protein database searching by the peptide-mass fingerprinting approach and MS/MS data suitable for protein database searching. Here the results of the two database-searching approaches are compared and the possibilities of combining the two approaches for rapid identification of protein are discussed. Also, we present a comparison of the results obtained using the three-position microfabricated device coupled to the ESI-QqTOFMS and to an ESI-ion trap MS. Finally the combination of C-terminal 18O labeling of peptides and the microfabricated system for automated combined peptide-mass fingerprinting and sequence-tag database searching is discussed.     

Activities of the enzymes participating in purine and free-radical metabolism in cancerous human colorectal tissues

Liquid chromatography-pneumatically assisted electrospray mass spectrometry with negative ionization has been used for the determination of acidic herbicides in ground water. Eighteen pesticides or pesticide degradation products belonging to several different groups of acidic herbicides (phenoxy acids, sulfonylureas, phenols, etc.) were covered in the study. Optimization of electrospray inlet conditions is described as well as results from investigations of the linearity of the detector response. Conditions for tandem mass spectrometry (MS-MS) detection of characteristic daughter ions formed by collision-induced dissociation (CID) of the parent ion are described and a comparison of obtainable instrument detection limits by single MS and MS-MS was made. Detection limits using MS in the selected ion monitoring (SIM) mode were generally in the order of 1 microgram/l or below, whereas detection limits were three-four times higher using MS-MS detection. A principle of analysis is proposed based on single quadrupole MS as a method for quantitative determination followed by verification of positive findings by CID MS-MS. Application of the method for detecting acidic herbicides residues in a "real-world" ground water sample is demonstrated.     

Analytical advances in detection of performance-enhancing compounds

The use and abuse of performance-enhancing substances has been an issue in sports since the ancient Greeks. The availability of numerous synthetic steroids and recombinant peptide hormones has made testing an analytical challenge. Recent advances in mass spectrometry have provided an opportunity to decrease detection limits. The Atlanta Olympic Games in 1996 marked the first time every specimen was screened by gas chromatography (GC) coupled to high-resolution mass spectrometry (MS). A further improvement may be seen with GC/MS/MS and quadrupole ion traps. Electrospray HPLC/MS has also been applied to the detection and confirmation of peptide hormones in urine. The ability to detect subtle differences in oligosaccharide structure may provide a way to detect abuse of recombinant glycoproteins. Simply decreasing detection limits is not enough; new technology also allows development of a foundation on which to base interpretation. Application of HPLC/MS/MS has allowed direct measurement of steroid conjugates in urine. The relative importance of sulfate, glucuronide, and other conjugates and metabolites of testosterone and epitestosterone can now be assessed. In the international sports arena, the impact of genetic metabolic disposition must also be considered if we are to provide an equitable system. Further research will establish more-refined criteria for the detection threshold of abused substances.