Michael W. Senko

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.

External Accumulation of Ions for Enhanced Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides for mass analysis of biological molecules with unrivaled mass accuracy, resolving power and sensitivity. However, ESI FTICR MS performance with on-line separation techniques such as liquid chromatography (LC) and capillary electrophoresis has to date been limited primarily by pulsed gas assisted accumulation and the incompatibility of the associated pump-down time with the frequent ion beam sampling requirement of on-line chromatographic separation. Here we describe numerous analytical advantages that accrue by trapping ions at high pressure in the first rf-only octupole of a dual octupole ion injection system before ion transfer to the ion trap in the center of the magnet for high performance mass analysis at low pressure. The new configuration improves the duty cycle for analysis of continuously generated ions, and is thus ideally suited for on-line chromatographic applications. LC/ESI FTICR MS is demonstrated on a mixture of 500 fmol of each of three peptides. Additional improvements include a fivefold increase in signal-to-noise ratio and resolving power compared to prior methods on our instrument.

Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions

The coupling of electrospray ionization with Fourier-transform mass spectrometry allows the analysis of large biomolecules with mass-measuring errors of less than 1 ppm. The large number of atoms incorporated in these molecules results in a low probability for the all-monoisotopic species. This produces the potential to misassign the number of heavy isotopes in a specific peak and make a mass error of ±1 Da, although the certainty of the measurement beyond the decimal place is greater than 0.1 Da. Statistical tests are used to compare the measured isotopic distribution with the distribution for a model molecule of the same average molecular mass, which allows the assignment of the monoisotopic mass, even in cases where the monoisotopic peak is absent from the spectrum. The statistical test produces error levels that are inversely proportional to the number of molecules in a distribution, which allows an estimation of the number of ions in the trapped ion cell. It has been determined, via this method that 128 charges are required to produce a signal-to-noise ratio of 3:1, which correlates well with previous experimental methods.

A High‐performance Modular Data System for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

The three major components of a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer include the vacuum system (including ion source), the magnet and a data system capable of performing the necessary instrument control for desired experiments. Most previous FTICR systems have used commercial data systems based on custom-built electronics controlled by proprietary mini-computers developed in the early 1980s. Here we present a high-performance data system based on a personal computer running user-friendly Windows software and readily available commercial components contained in a VXI chassis and a minimal complement of simple custom electronics. The system uses a VXI pattern generator to control all aspects of the experiment. The flexibility of the pattern generator allows for performance of all current FTICR experimental sequences.

Electrospray Ionization Fourier Transform Ion Cyclotron Resonance at 9.4 T

We present the first results from a new electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer operated at a magnetic field of 9.4 T (i.e. > or = 2.4 T higher than for any prior FTICR instrument). The 9.4 T instrument provides substantially improved performance for large molecules (> or = 50% increase in mass resolving power) and complex mixtures (> or = 100% increase in dynamic range) compared to lower-field (< or = 6 T) instruments. The higher magnetic field makes possible larger trapped-ion population without introduction of significant space--charge effects such as spectral peak shift and/or distortion, and coalescence of closely-spaced resonances. For bovine ubiquitin (8.6 kDa) we observe accurate relative isotopic abundances at a signal-to-noise ratio greater than 1000:1, whereas a complete nozzle-skimmer dissociation electrospray ionization (ESI) FTICR mass spectrum of bovine carbonic anhydrase (29 kDa) is achieved from a single scan with a signal-to-noise ratio of more than 250:1. Finally, we are able to obtain mass resolving power, m/delta m > 200,000, routinely for porcine serum albumin (67 kDa). The present performance guides further modifications of the instrument, which should lead to significant further improvements.

High-performance mass spectrometry: Fourier transform ion cyclotron resonance at 14.5 Tesla.

We describe the design and current performance of a 14.5 T hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer. Ion masses are routinely determined at 4-fold better mass accuracy and 2-fold higher resolving power than similar 7 T systems at the same scan rate. The combination of high magnetic field and strict control of the number of trapped ions results in external calibration broadband mass accuracy typically less than 300 ppb rms, and a resolving power of 200,000 (m/Delta m50% at m/z 400) is achieved at greater than 1 mass spectrum per second. Novel ion storage optics and methodology increase the maximum number of ions that can be delivered to the FTICR cell, thereby improving dynamic range for tandem mass spectrometry and complex mixture applications.

Fourier-transform electrospray instrumentation for tandem high-resolution mass spectrometry of large molecules

Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York, USA Mass spectrometry instrumentation providing unit resolution and lo-ppm mass accuracy for molecules larger than 10 kDa was first reported in 1991. This instrumentation has now been improved with a 6.2-T magnet replacing that of 2.8 T, a more efficient vacuum system, ion injection with controlled ion kinetic energies, accumulated ion trapping with an open-cylindrical ion cell, acquisition of 2M data points, and updated electrospray apparatus. The resulting capabilities include resolving power of 5 × 105 for a 29-kDa protein, less than l-ppm mass measuring error, and dissociation of protein molecular ions to produce dozens of fragment ions whose exact masses can be identified from their mass-to-charge ratio values and isotopic peak spacing.

Automated assignment of charge states from resolved isotopic peaks for multiply charged ions

The recent proliferation of electrospray as an ionization method has greatly increased the ability to perform analyses of large biomolecules by using mass spectrometry. The major advantage of electrospray is the ability to produce multiply charged ions, which brings large molecules down to a mass-to-charge ratio range amenable to most instruments. Multiple charging is also a disadvantage because mass (m) becomes ambiguous unless charge (z) can be assigned. This is typically performed with simple algorithms that use multiple peaks of the same m and different z, but these methods are difficult to apply to complex mixtures and not applicable when only one z appears for each m. The use of mass analyzers with higher resolving powers, like the Fourier transform mass spectrometer, allows resolution of isotopic peaks, providing an internal 1-Da mass scale that can be used for unambiguous charge assignment. Manual assignment of charge state from the isotopic peaks is time consuming and becomes inaccurate when either the signal level or resolving power are low. For these cases, computer algorithms based on pattern recognition techniques have been developed to assist in assignment of charge states to isotopic clusters. These routines provide for more rapid analysis with higher accuracy than available manually.

Novel Parallelized Quadrupole/Linear Ion Trap/Orbitrap Tribrid Mass Spectrometer Improving Proteome Coverage and Peptide Identification Rates

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general experimental throughput.