A dynamic ion cooling technique for FTICR mass spectrometry.

A fast dynamic ion cooling technique based upon the adiabatic invariant phenomenon for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is presented. The method cools ions in the FTICR trap more efficiently, within a few hundred milliseconds without the use of a buffer gas, and results in a substantial signal enhancement. All performance aspects of the FTICR spectrum, e.g., peak intensities, mass resolution, and mass accuracy, improve significantly compared with cooling based on ion-ion interactions. The method may be useful in biological applications of FTICR, such as in proteomic studies involving extended on-line liquid chromatography (LC) separations, in which both the duty cycle and mass accuracy are crucially important.

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry.

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry.

The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.

Electrospray ionization-Fourier transform ion cyclotron mass spectrometry using ion preselection and external accumulation for ultrahigh sensitivity.

The dynamic range of Fourier transform ion cyclotron mass spectrometry (FTICR) is typically limited by the useful charge capacity of an FTICR cell (to approximately 10(6) to 10(7) elementary charges) and the minimum number of ions required to produce a useful signal (approximately 10(2) elementary charges). We show that the expansion of the dynamic range by 2 orders of magnitude can be achieved by preselecting lower abundance species in a quadrupole interface to an electrospray ionization (ESI) source. Ion preselection is then followed by ion accumulation in external to the FTICR cell a linear (2-D) quadrupole trap and subsequent transfer to the region of high magnetic field for gated trapping in the FTICR cell. Two modes of ion preselection, using either the quadrupole filtering mode or rf-only dipolar excitation, were studied and mass resolutions of 30 to 100 were achieved for selective external ion accumulation of peptides and proteins with molecular weights ranging from 500 to 17,000 Da. The ability to selectively eject the most abundant species before trapping in the FTICR has enormous practical benefits for increasing the sensitivity and dynamic range of measurements.

Design and implementation of a new electrodynamic ion funnel.

A new electrodynamic (rf) ion funnel has been developed and evaluated for use in the interface regions (at approximately 1-10 Torr) of atmospheric pressure ion sources (e.g., electrospray ionization (ESI) for mass spectrometry). The ion funnel consists of a ring electrode ion guide with decreasing i.d. and with a superimposed dc potential gradient along the ring stack. The thicknesses of the ring electrodes and the spacings between them were reduced to 0.5 mm from 1.59 mm compared to those used for previous designs. The new ion funnel displays a significant improvement in low-mass transmission (m/z >200) and sensitivity compared to previous designs. The transmission efficiencies for electrosprayed peptides and proteins (ranging in mass from 200 to 17,000 Da) were typically 50-60% of total incoming currents from a heated capillary inlet. The transmitted ion currents were a factor of 30-56 greater than those of the standard interface for peptide samples and a factor of 18-22 greater than those for protein samples. The sensitivity gains realized at the MS detector were somewhat lower, possibly due to space charge effects in the octapole ion beam guide following the ion funnel. The improved ion transmission properties result primarily from the use of reduced spacings between ring electrodes. We also show that the ion funnel can be operated in two different modes, one using low-rf-amplitude scans, allowing fragile noncovalent complexes (as well as generally undesired adducts) to be transmitted, and the other using high-rf-amplitude scans, providing greater collisional activation and more effective adduct removal (or the dissociation of lower m/z species).

Accurate mass multiplexed tandem mass spectrometry for high-throughput polypeptide identification from mixtures.

We report a new tandem mass spectrometric approach for the improved identification of polypeptides from mixtures (e.g., using genomic databases). The approach involves the dissociation of several species simultaneously in a single experiment and provides both increased speed and sensitivity. The data analysis makes use of the known fragmentation pathways for polypeptides and highly accurate mass measurements for both the set of parent polypeptides and their fragments. The accurate mass information makes it possible to attribute most fragments to a specific parent species. We provide an initial demonstration of this multiplexed tandem MS approach using an FTICR mass spectrometer with a mixture of seven polypeptides dissociated using infrared irradiation from a CO2 laser. The peptides were added to, and then successfully identified from, the largest genomic database yet available (C. elegans), which is equivalent in complexity to that for a specific differentiated mammalian cell type. Additionally, since only a few enzymatic fragments are necessary to unambiguously identify a protein from an appropriate database, it is anticipated that the multiplexed MS/MS method will allow the more rapid identification of complex protein mixtures with on-line separation of their enzymatically produced polypeptides.

Initial implementation of an electrodynamic ion funnel with Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has become a widely used method to study biopolymers. The method, in combination with an electrospray ionization (ESI) source has demonstrated the highest resolution and accuracy yet achieved for characterization of biomolecules and their noncovalent complexes. The most common design for the ESI interface includes a heated capillary inlet followed by a skimmer having a small orifice to limit gas conductance between a higher pressure (1 to 5 torr) source region and the lower pressure ion guide. The ion losses in the capillary-skimmer interface are large (estimated to be more than 90%) and thus reduce achievable sensitivity. In this work, we report on the initial implementation of a newly developed electrodynamic ion funnel in a 3.5 tesla ESI-FTICR mass spectrometer. The initial results show dramatically improved ion transmission as compared to the conventional capillary-skimmer arrangement. An estimated detection limit of 30 zeptomoles (approximately 18,000 molecules) has been achieved for the analysis of the proteins with molecular weights ranging from 8 to 20 kDa.

High-mass-measurement accuracy and 100% sequence coverage of enzymatically digested bovine serum albumin from an ESI-FTICR mass spectrum.

The application of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to the analysis of polypeptide mixtures resulting from proteolytic digestion is described. A new 11.5-T FTICR mass spectrometer has been applied for the analysis of tryptic digestion mixtures of the protein bovine serum albumin (BSA). The improved cyclotron frequency stability and reduced frequency shifts observed over a wide range of trapped ion population sizes provide the ability to signal average spectra without degrading mass measurement accuracy, requiring internal calibration or advanced data processing schemes to compensate for variations in ion cyclotron signals brought about by different population sizes. A total of 100 spectra were signal-averaged leading to the observation of a total of 123 isotope distributions with a signal-to-noise ratio greater than 3:1. From those distributions, 86 can be ascribed to tryptic fragments of BSA on the basis of mass measurement errors of 10 ppm or less. Of these, 71 were within 2 ppm error limits corresponding to complete amino acid sequence coverage and an average error of 0.77 ppm. These results indicate that high-accuracy measurements are feasible for a large number of species detected simultaneously without the necessity for internal calibration and indicate the potential of such measurements, when combined with chromatographic separations, for facilitating more rapid identification of large numbers of proteins.