Jae C. Schwartz

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 MSn 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.

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.

Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s—increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

High resolution on a quadrupole ion trap mass spectrometer

By using a modified ion trap mass spectrometer, resolution in excess of 30,000 (FWHM) at m I z 502 is demonstrated. The method of increasing resolution in the ion trap mass spectrometer operated in the mass-selective instability mode depends on decreasing the rate of scanning the primary radio frequency amplitude as well as using resonance ejection at the appropriate frequency and amplitude. A theoretical basis for the method is introduced.

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 bakers 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.

Automated strategies for obtaining standardized collisionally induced dissociation spectra on a benchtop ion trap mass spectrometer

An automated method for obtaining standardized collisionally induced dissociation (CID) spectra using two novel ion activation techniques on a quadrupole ion trap mass spectrometer is described. This strategy simultaneously produces optimal CID spectra “on-the-fly” and maximizes the amount of structurally specific fragment ions obtained in a single experiment, thus eliminating the need for individual tuning of specific mass-to-charge ratios and permitting fragmentation conditions to be more consistently reproduced. Copyright

Infrared Photoactivation Reduces Peptide Folding and Hydrogen-Atom Migration following ETD Tandem Mass Spectrometry

Electron capture dissociation (ECD)[1] results from the mutual storage of thermal electrons with multiply protonated peptide cations – an experiment generally performed within the high magnetic field of a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS). The technique is particularly useful as it generates random backbone cleavage with little regard to the presence of post-translational modifications (PTMs), amino acid composition, or peptide length. Electron transfer dissociation (ETD),[2] the ion-ion analogue of ECD, is conducted in radio frequency (RF) quadrupole ion trap devices where radical anions serve as electron donors. Because it can be implemented on virtually any mass spectrometer with an RF ion transfer or storage device, ETD has become an increasingly widespread dissociation method.

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.

QUADRUPOLE ION TRAP MASS SPECTROMETRY

A number of other features of ITMS systems that will enhance their ability to analyze biological macromolecules are worth mentioning. As has already been demonstrated for ESI/quadrupole, ESI/magnetic sector, and ESI/FTICR systems, the capability of inducing fragmentation of the ESI-generated multiply charged ions of biological macromolecules in the capillary/skimmer region of the ESI source and subsequently selectively analyzing fragments can also be carried out with the QITMS, as we have demonstrated using bovine serum albumin (data not shown). The ability to carry out chemical reactions on biological macromolecules inside the QITMS has been demonstrated by McCluckey et al. by showing that the introduction of a pulse of volatile base, such as diethylamine, can result in proton removal from multiply charged protein ions, resulting in species with lower charge states. The application of the technique of deuterium exchange of active hydrogens on peptides to simplify the interpretation of MS/MS sequencing experiments can be implemented for ESI/QITMS. Carrying out such exchange inside the ITMS may also be possible, with resulting analytical advantages. Reports of a hybrid QITMS-TOF system, which was operated with either ESI or MALDI methodology, and which demonstrated low femtomolar sensitivity with higher resolution of the TOF analyzer because of ion injection of essentially monoenergetic ions from the QITMS into the TOF, illustrate additional uses of the QITMS. The reverse combination (e.g., ESI/TOF/QITMS or MALDI/TOF/QITMS) could afford preselection of ions for even higher performance in the QITMS, because space charging (loss of performance such as resolution because of too much charge in close proximity in the ion trap) would be minimized. Opportunities for the application of QITMS technology for the analysis of biological macromolecules abound, including ultrahigh-sensitivity protein sequencing using specifically derivatized amino acids released by Edman chemistry; rapid sequencing of MHC-associated antigenic peptides of variable length (approximately nonamers for the MHC I complexes to > dodecamers for the MHC II complexes), which are available in only very low amounts (femtomole/attomole) and in very complex mixtures (5000-10,000 species) of closely related peptide structures; ultrahigh-sensitivity analysis of peptides and proteins directly in vivo using microelectrospray; direct analysis of metal ion binding to peptides and proteins and analysis of noncovalent interactions, including conformation; and possible analysis of plasmid DNA, as has been suggested by ESI ionization of a 2-MDa DNA species. In summary, the ability of the QITMS to interface to key separations systems such as HPLC and HPCE through the critical ionization techniques of ESI and MALDI, coupled with the high mass range, high mass resolution, high sensitivity, high-efficiency CID, and MS capabilities of this device, will provide an astonishing array of cost-effective capabilities for the qualitative and quantitative analysis of biological macromolecules.

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.