Enhanced Sequence Coverage of Large Proteins by Combining Ultraviolet Photodissociation with Proton Transfer Reactions.

Ultraviolet photodissociation (UVPD) produces rich and informative fragmentation of intact protein ions, but in the case of high mass proteins (>30 kDa) the spectra are congested with overlapping isotope patterns of highly charged fragment ions. In the most congested regions, many fragments cannot be confidently identified even when high-resolution mass analyzers and modern deconvolution algorithms are used. Gas-phase ion-ion proton transfer reactions (PTR), which reduce the charge states of highly charged ions, can be used to alleviate this congestion and facilitate the identification of additional fragment ions when performed following UVPD. We have developed protocols for sequentially performing PTR on multiple populations of ions generated by UVPD in a way that can be tailored to balance the depth of characterization with speed and throughput. The improvements in sequence coverage and fragment identifications are demonstrated for four proteins ranging in size from 29 to 56 kDa. Sequence coverages up to 80% were achieved for carbonic anhydrase (29 kDa), 50% for aldolase (39 kDa), 46% for enolase (46 kDa), and 27% for glutamate dehydrogenase (56 kDa), and up to 74% sequence coverage was obtained for 25 kDa antibody drug conjugate subunits in online LC-MS experiments.

Implementation of Fragment Ion Protection (FIP) during Ultraviolet Photodissociation (UVPD) Mass Spectrometry.

Ultraviolet photodissociation (UVPD) is a nonselective activation method in which both precursor and fragment ions may absorb photons and dissociate. Photoactivation of fragment ions may result in secondary or multiple generations of dissociation, which decreases the signal-to-noise ratio (S/N) of larger fragment ions owing to the prevalent subdivision of the ion current into many smaller, often less informative, fragment ions. Here we report the use of dipolar excitation waveforms to displace fragment ions out of the laser beam path, thus alleviating the extent of secondary dissociation during 193 nm UVPD. This fragment ion protection (FIP) strategy increases S/N of larger fragment ions and improves the sequence coverage obtained for proteins via retaining information deeper into the midsection of protein sequences.

Ultraviolet Photodissociation Induced by Light-Emitting Diodes in a Planar Ion Trap.

The first application of light-emitting diodes (LEDs) for ultraviolet photodissociation (UVPD) mass spectrometry is reported. LEDs provide a compact, low cost light source and have been incorporated directly into the trapping cell of an Orbitrap mass spectrometer. MS/MS efficiencies of over 50 % were obtained using an extended irradiation period, and UVPD was optimized by modulating the ion trapping parameters to maximize the overlap between the ion cloud and the irradiation volume.

A differentially pumped dual linear quadrupole ion trap (DLQIT) mass spectrometer: a mass spectrometer capable of MS(n) experiments free from interfering reactions.

A novel differentially pumped dual linear quadrupole ion trap (DLQIT) mass spectrometer was designed and built to facilitate tandem MS experiments free from interfering reactions. The instrument consists of two differentially pumped Thermo Scientific linear quadrupole ion trap (LQIT) systems that have been connected via an ion transfer octupole encased in a machined manifold. Tandem MS experiments can be performed in the front trap and then the resulting product ions can be transferred via axial ejection into the back trap for further, independent tandem MS experiments in a differentially pumped area. This approach allows the examination of consecutive collision-activated dissociation (CAD) and ion-molecule reactions without unwanted side reactions that often occur when CAD and ion-molecule reactions are examined in the same space. Hence, it greatly facilitates investigations of ion structures. In addition, the overall lower pressure of the DLQIT, as compared to commercial LQIT instruments, results in a reduction of unwanted side reactions with atmospheric contaminants, such as water and oxygen, in CAD and ion-molecule experiments.

A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.

Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3-5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.

Activated-ion electron transfer dissociation improves the ability of electron transfer dissociation to identify peptides in a complex mixture.

Using a modified electron transfer dissociation (ETD)-enabled quadrupole linear ion trap (QLT) mass spectrometer, we demonstrate the utility of IR activation concomitant with ETD ion-ion reactions (activated-ion ETD, AI-ETD). Analyzing 12 strong cation exchanged (SCX) fractions of a LysC digest of human cell protein extract using ETD, collision-activated dissociation (CAD), and AI-ETD, we find that AI-ETD generates 13 405 peptide spectral matches (PSMs) at a 1% false-discovery rate (1% FDR), surpassing both ETD (7 968) and CAD (10 904). We also analyze 12 SCX fractions of a tryptic digest of human cell protein extract and find that ETD produces 6 234 PSMs, AI-ETD 9 130 PSMs, and CAD 15 209 PSMs. Compared to ETD with supplemental collisional activation (ETcaD), AI-ETD generates ∼80% more PSMs for the whole cell lysate digested with trypsin and ∼50% more PSMs for the whole cell lysate digested with LysC.

Development of a linear ion trap/orthogonal-time-of-flight mass spectrometer for time-dependent observation of product ions by ultraviolet photodissociation of peptide ions.

A hybrid linear ion trap/orthogonal time-of-flight (TOF) mass spectrometer has been developed to observe time-dependent vacuum ultraviolet photodissociation product ions. In this apparatus, a reflectron TOF mass analyzer is orthogonally interfaced to an LTQ using rf-only octopole and dc quadrupole ion guides. Precursor ions are generated by electrospray ionization and isolated in the ion trap. Subsequently they are directed to the TOF source where photodissociation occurs and product ions are extracted for mass analysis. To detect photodissociation product ions having axially divergent trajectories, a large rectangular detector is utilized. With variation of the time between photodissociation and orthogonal extraction in the TOF source, product ions formed over a range of times after photoexcitation can be sampled. Time-dependent observation of product ions following 157 nm photodissociation of a singly charged tryptic peptide ion (NWDAGFGR) showed that prompt photofragment ions (x- and v-type ions) dominate the tandem mass spectrum up to 1 micros after the laser shot, but the intensities of low energy thermal fragment ions (y-type ions) become comparable several microseconds later. Different proton mobilization time scales were observed for arginine- and lysine-terminated tryptic peptides.

Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures.

The considerable progress in high-throughput proteomics analysis via liquid chromatography-electrospray ionization-tandem mass spectrometry over the past decade has been fueled to a large degree by continuous improvements in instrumentation. High-throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become broadly adopted analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ Velos) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times 2 times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. The greatly increased ion flux into the mass spectrometer in combination with improved isolation of low-abundance precursor ions resulted in increased detection of low-abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker's yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Alternatively, faster cycle times on the new instrument allowed for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. When mass analysis was carried out with resolution in excess of 25,000 full width at half-maximum (fwhm), it became possible to isotopically resolve a small intact protein and its fragments, opening possibilities for top down experiments.

Top-down protein fragmentation by infrared multiphoton dissociation in a dual pressure linear ion trap.

Infrared multiphoton dissociation (IRMPD) was implemented in a novel dual pressure linear ion trap for rapid top-down proteomics. The high pressure cell provided improved trapping and isolation efficiencies while the isotopic profiles of 10+ charged ions could be resolved by mass analysis in the low pressure cell that enabled effective top down protein identification. Striking differences between IRMPD in the low pressure cell and CID in the high pressure cell were observed for proteins ranging from 8.6 to 29 kDa. Because of secondary dissociation, IRMPD yielded product ions in significantly lower charge states as compared to CID, thus facilitating more accurate mass identification and streamlining product ion assignment. This outcome was especially useful for database searching of larger proteins (approximately 29 kDa) as IRMPD substantially improved protein identification and scoring confidence. Also, IRMPD showed an increased selectivity toward backbone cleavages N-terminal to proline and C-terminal to acidic residues (especially for the lowest charge states), which could be useful for a priori spectral predictions and enhanced database searching for protein identification.

Infrared multiphoton dissociation of peptide cations in a dual pressure linear ion trap mass spectrometer.

A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells-the first a high pressure cell operated at nominally 5 x 10(-3) Torr and the second a low pressure cell operated at nominally 3 x 10(-4) Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y(1) fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of approximately 100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra.

A two-dimensional quadrupole ion trap mass spectrometer.

The use of a linear or two-dimensional (2-D) quadrupole ion trap as a high performance mass spectrometer is demonstrated. Mass analysis is performed by ejecting ions out a slot in one of the rods using the mass selective instability mode of operation. Resonance ejection and excitation are utilized to enhance mass analysis and to allow isolation and activation of ions for MS(n) capability. Improved trapping efficiency and increased ion capacity are observed relative to a three-dimensional (3-D) ion trap with similar mass range. Mass resolution comparable to 3-D traps is readily achieved, including high resolution at slower scan rates, although adequate mechanical tolerance of the trap structure is a requirement. Additional advantages of 2-D over 3-D ion traps are also discussed and demonstrated.

Activated ion ETD performed in a modified collision cell on a hybrid QLT-Oribtrap mass spectrometer.

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO2 laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety's high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.

Multipurpose dissociation cell for enhanced ETD of intact protein species.

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell's longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

MS(M), an efficient workflow for metabolite identification using hybrid linear ion trap Orbitrap mass spectrometer.

Identification of drug metabolites can often yield important information regarding clearance mechanism, pharmacologic activity, or toxicity for drug candidate molecules. Additionally, the identification of metabolites can provide beneficial structure-activity insight to help guide lead optimization efforts towards molecules with optimal metabolic profiles. There are challenges associated with detecting and identifying metabolites in the presence of complex biological matrices, and new LC-MS technologies have been developed to meet these challenges. In this report, we describe the development of an experimental approach that applies unique features of the hybrid linear ion trap Orbitrap mass spectrometer to streamline in vitro and in vivo metabolite identification experiments. The approach, referred to as MS(M), utilizes multiple collision cells, dissociation methods, mass analyzers, and detectors. With multiple scan types and different dissociation modes built into one experimental method, along with flexible post-acquisition analysis options, the MS(M) workflow offers an attractive option to fast and reliable identification of metabolites in different kinds of in vitro and in vivo samples. The MS(M) workflow was successfully applied to metabolite identification analysis of verapamil in both in vitro rat hepatocyte incubations and in vivo rat bile samples.

Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors.

Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).

Rectilinear ion trap mass spectrometer with atmospheric pressure interface and electrospray ionization source.

A rectilinear ion trap (RIT) mass analyzer was incorporated into a mass spectrometer fitted with an electrospray ionization source and an atmospheric pressure interface. The RIT mass spectrometer, which was assembled in two different configurations, was used for the study of biological compounds, for which performance data are given. A variety of techniques, including the use of a balanced rf, elevated background gas pressure, automatic gain control, and resonance ejection waveforms with dynamically adjusted amplitude, were applied to enhance performance. The capabilities of the instrument were characterized using proteins, peptides, and pharmaceutical drugs. Unit resolution and an accuracy of better than m/z 0.2 was achieved for mass-to-charge (m/z) ratios up to 2000 Th at a scan rate of approximately 3000 amu/(charge.s) while reduced scan rates gave greater resolution and peak widths of less than m/z 0.5 over the same range. The mass discrimination in trapping externally generated ions was characterized over the range m/z 190-2000 and an optimized low mass cutoff value of m/z 120-140 was found to give equal trapping efficiencies over the entire range. The radial detection efficiency was measured as a function of m/z ratio and found to rise from 35% at low m/z values to more than 90% for ions of m/z 1800. The way in which the ion trapping capacity depends on the dc trapping potential was investigated by measuring the mass shift due to space charge effects, and it was shown that low trapping potentials minimize space charge effects by increasing the useful volume of the device. The collision-induced dissociation (CID) capabilities of the RIT instrument were evaluated by measuring isolation efficiency as a function of mass resolution as well as measuring peptide CID efficiencies. Overall CID efficiencies of more than 60% were easily reached, while isolation of an ion with unit resolution at m/z 524 was achieved with high rejection (>95%) of the adjacent ions. The overall analytical capabilities of the ESI-RIT instrument were demonstrated with the analysis of a mixture of pharmaceutical compounds using multiple-stage mass spectrometry.

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).

Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications.

We describe the design and performance of a prototype high performance hybrid mass spectrometer. This instrument consists of a linear quadrupole ion trap (QLT) coupled to a Fourier transform ion cyclotron resonance mass analyzer (FTMS). This configuration provides rapid and automated MS and MS/MS analyses, similar to the "data dependent scanning" found on standard 3-D Paul traps, but with substantially improved internal scan dynamic range, mass measurement accuracy, mass resolution, and detection limits. Sequence analysis of peptides at the zeptomole level is described. The recently released, commercial version of this instrument operates in the LC/MS mode (1 s/scan) with a mass resolution of 100 000 and is equipped with automatic gain control to provide mass measurement accuracy of 1-2 ppm without internal standard. Methodology is described that uses this instrument to compare the post-translational modifications present on histone H3 isolated from asynchronously growing cells and cells arrested in mitosis.